Species diversity and molecular taxonomy of symbiotic crustaceans on Portunus pelagicus (Linnaeus, 1758) in Vietnam, with remarks on host records and morphological variation

Abstract

The blue swimming crab (Portunus pelagicus (Linnaeus, 1758)) is an economically and ecologically important species in Vietnam, and a potential subject for aquaculture as well. More than 400 individual crabs were collected along the Vietnamese coastline and examined for ectosymbiotic crustaceans. Two molecular markers (28S rRNA and COI mtDNA) were applied for species delineation. Seven species were reported and described, of which six are cirripede barnacles (Thecostraca, Thoracia); and one parasitic copepod Choniosphaera indica Gnanamuthu, 1954 (Copepoda, Podoplea). Four species (Chelonibia testudinaria (Linnaeus, 1758), Semibalanus sp., Octolasmis neptuni (MacDonald, 1869), and Dianajonesia tridens (Aurivillius, 1894)) were the first records for both host and location. The symbiotic crustaceans occupy specific niches on the crab body, and vary in their infestation status. Molecular taxonomy of symbiotic crustaceans was classified and confirmed based on sequence similarity and phylogenetic analyses.

Keywords:
Crustaceans; infestation; phylogeny; Portunidae; symbionts

INTRODUCTION

The swimming crab, Portunus pelagicus (Linnaeus, 1758) is a major economic species throughout the Indo-Pacific and to the coast of Africa (Galil and Innocenti, 1999). In Vietnam, it is widely distributed throughout the coastal waters and nearby islands (Vu et al., 2014). Throughout its native range, it is a valued market organism with numerous reports of its commercial value as a multi-million dollar export commodity between Vietnam and Japan and the United States of America (VASEP, 2021). Crabs play ecological important roles, through complex food webs, in coastal and marine ecosystems, especially in mangrove forests, seagrass beds and coral reefs (Kunsook et al., 2014a; 2014b). Additionally, their carbonate carapaces are widely known as a living substrate for many epibiont/symbiont organisms (Abelló and Corbera, 1996; Gaddes and Sumpton, 2004; Dvoretsky, 2012; Machado et al., 2013).

Reciprocal selection pressure between the host and its symbiotic species can potentially alter species diversity, ecological function, and community dynamics (Galil and Innocenti, 1999). The diversity of the symbiotic community on P. pelagicus is known to depend on its geographic distribution (Shields, 1992; Shields and Wood, 1993). Among the symbiotic crustacean species, the barnacles frequently found attached to decapod crustaceans have received the most attention. Their symbiotic association depends on the host’s biological characteristics, such as distribution, sex, maturity stage, molt cycle, and size (Weng, 1987; Gaddes and Sumpton, 2004; Klinbunga et al., 2007; Babu et al., 2012; Machado et al., 2013). Growing numbers of symbiotic species have been reported on portunid decapod species (Hudson and Lester, 1994; Isaeva et al., 2005; Babu et al., 2012). In Vietnam, several symbiotic species have so far been detected on P. pelagicus and Portunus trituberculatus (Miers, 1876). Among these, two species (Carcinonemertes mitsukuriiTakakura, 1910 and Choniosphaera indicaGnanamuthu, 1954), recognized egg eating parasites, are known to have a negative impact on host populations (Vo et al., 2013; Le et al., 2018a; 2018b).

In the past decade, molecular markers have been increasingly applied to investigate species diversity and phylogenetic relationships, including research examining the phylogenetic position and evolution of Cirripedia (Mizrahi et al., 1998; Wu, 2011; Kwiatkowski et al., 2012; Yusa et al., 2012; Filipiak et al., 2016). So far, no research has focused on elucidating the phylogenetic relationships of the symbiotic crustacean community on the blue swimming crab.

The current research conducts the first comprehensive study of species diversity, infestation status, and molecular taxonomy of symbiotic crustacean associations occurring on P. pelagicus distributed along the Vietnamese coastline.

MATERIAL AND METHODS

Symbiotic crustacean sampling, identification, and infestation status

Blue swimming crabs (P. pelagicus) were collected from the Vietnamese coastline from Cat Ba Island - Hai Phong, Ha Long Bay - Quang Ninh in the North; Nha Trang and Van Phong Bay - Khanh Hoa, Song Cau and Tuy Hoa - Phu Yen in the Center, and Phu Quoc Island and Rach Gia - Kien Giang in the South. The crabs were transported alive in aerated sea water to the laboratory where they were kept in aquaria until dissected. Sampling information of crab individuals collected is presented in Tab. 1.

Table 1.
Sampling site information and number of individuals of Portunus pelagicus collected along the Vietnam coastline.

Ecoregion	Sampling location	Longitude	Latitude	No. of Individuals
North	Cat Ba Island - Hai Phong	107°00’57.25”E	20°45’01.745”N	91
	Ha Long Bay - Quang Ninh	107°04’59.42”E	20°56’55.43”N	58
Central	Nha Trang and Van Phong Bay - Khanh Hoa	109°13’02.39”E 109°21’31.60”E	12°12’46.85”N 12°29’40.12”N	100
	Song Cau and Tuy Hoa - Phu Yen	109°13’46.54”E 109°18’48.98”E	13°27’36.21”N 13°05’02.596”N	100
South	Phu Quoc Island - Kien Giang	103°57’11.07”E	10°13’23.06”N	41
	Rach Gia - Kien Giang	105° 04’28.85”E	90°59’23.39”N	89

Crabs were examined externally for symbiotic crustaceans. Each crab was divided into 6 separate parts: (1) Carapace; (2) Limbs (maxillipeds, chelipeds, and swimming legs); (3) Abdomen, including egg clutches (if any); (4) Mouth parts; (5) Gills; and (6) Sternum (see ^Appendix, Fig. A1). Each part was placed separately into petri dishes containing clean seawater for inspection by the naked eye and a stereoscope (Olympus SZX9).

Freshly collected symbiotic crustacean species were used for the descriptions and identification. The individuals intended as whole-mounts were transferred to a vial of 70 % alcohol, and those for DNA analysis were preserved in 95 % EtOH, and stored at -20 °C. Symbiotic crustaceans were identified using various keys and species descriptions follow Jeffries and Voris (1996); Jeffries et al. (2005); Cheang et al. (2013). Infestation status of symbiotic species were examined by prevalence and mean intensity, as defined in Margolis et al. (1982) and Rózsa et al. (2000).

Molecular taxonomy

DNA was extracted from individuals of each collected symbiont species using DNeasy Tissue Extraction Kit (Qiagen) in accordance with the manufacturer’s instructions. 28S rRNA and COI (Cytochrome c oxidase subunit I) mitochondrial DNA were amplified using primer LSU 5 and 1500R (Olson et al., 2003), and LCO1490 and HCO2198 (Folmer et al., 1994), respectively.

PCR reactions were performed using a total volume of 50 µl with components at the following concentrations: 10 µL of DreamTaq buffer 10X (Thermo Fisher Scientific), 2 µL dNTP (10 mM), 2 µL each primer (10 mM), 1.25 unit of DreamTaq polymerase (5U/µl), 5 µl DNA template and distilled water to the final volume. Amplification was implemented using the following PCR profile: a preliminary denaturation at 94 °C for 3 minutes (min), followed by 35 cycles of 94 °C for 45 seconds (s), annealing for 45 s (for 28S rRNA, COI mtDNA genes at 56 °C, 42 °C, respectively), and then 72 °C for 45 s. This was followed by a final extension period at 72 °C for 7 min before the samples were cooled to 4 °C. PCR products were run on a 1.5 % agarose gel for confirmation of equal length against an appropriate size marker. The PCR products were purified using DNA purification kits (Promega) and pre-sequenced using dye- labels dideoxy terminator (Big Dye Terminator 3.1, Applied Biosystems) with the same primer as the PCR reactions at the following temperatures: 96 °C for 30 s, 50 °C for 30 s and 60 °C for 4 min. Sequences of both strands were generated on an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems) using the amplification primers.

Sequence contigs were assembled using the Geneious Pro 5.5.7 (Kearse et al., 2012). The resulting sequences were confirmed by the Basic Local Alignment Search Tool (BLAST, https://blast.ncbi.nlm.nih.gov/Blast.cgi). The sequences were aligned, analyzed using BioEdit 7.0.5.3 (Hall, 1999), and submitted to GenBank. Sequence identity matrix was used to investigate similarity/identity values. Information on gene application, and Genbank accession numbers are presented in Tab. 2.

Table 2.
Species list, infestation status (Portunus pelagicus, n = 479), and Genbank accession numbers for all sequences of symbiotic crustaceans used in the phylogenetic analysis. (1) Carapace, (2) Limbs (maxilliped, cheliped, and swimming legs), (3) Abdomen, including egg clutches (if any), (4) Mouth parts, (5) Gills, and (6) Sternum. * - sequences from Genbank; x - sequences not available; NI: no information

Species	Prevalence Mean Intensity	Infection sites	COI mtDNA	28S rRNA	Host/substrata
I. Class: Thecostraca, Subclass: Cirripedia, Infraclass: Thoracica
I.1. Order: Lepadiformes
Octolasmis angulata	75.57 57.26±93.06	(2), (3), (5)	MH753551	MH727736	Swimming crab (Portunus pelagicus)
Octolasmis neptuni	11.48 21.55±30.26	(2), (3), (5)	MK541906	MH727737	Swimming crab (Portunus pelagicus)
Octolasmis warwicki	15.45 23±31.12	(1), (2), (3), (4)	MH753552	MH727739	Swimming crab (Portunus pelagicus)
Octolasmis cor*			KC138499	EU082326	NI
Octolasmis sp.*			x	EU082327	NI
Octolasmis unguisiformis*			LC467960	LC467957	Sea urchin (Echinothrix diadema)
Dianajonesia tridens	19.00 7.95±8.49	(2), (3), (4), (5)	MH753553	MH727738	Swimming crab (Portunus pelagicus)
I.2. Order: Sessilia
Chelonibia testudinaria	14.82 9.32±9.96	(1), (2), (6)	MH753554	MK087648	Swimming crab (Portunus pelagicus)
Chelonibia testudinaria*			KF042514	AB723914	Green sea turtle (Chelonia mydas)
Chelonibia testudinaria*			KF042515	KM217527	Hawksbill sea turtle (Eretmochelys imbricate)
Chelonibia patula*			JF823674	EU082295	Mud crab (Scylla serrata)
Chelonibia manati* (= C. testudinaria)			JN589813	AB723917	West Indian manatee(Trichechus manatus)
Chelonibia caretta*			KF042512	AB723915	Hawksbill sea turtle (Eretmochelys imbricate)
Chelonibia caretta*			KF042513	KM217526	Loggerhead Sea turtle (Caretta caretta)
Semibalanus cariosus*			KM611728	AY520593	NI
S. balanoides*			MF748337	EU370440	NI
Semibalanus sp.	0.21/6	(1)	MH753555	MH727740	Swimming crab (Portunus pelagicus)
Sacculina carcini*			KT209175	AY520622	NI
II. Class: Copepoda, Superorder: Podoplea
II.1. Order: Siphonostomatoida
Choniosphaera indica	2.09 20.90±4.97	(3)	MK541907	MK087649	Swimming crab (Portunus pelagicus)
Asterocheres lilljeborgi*			KR049050	KR048868	NI
Parabrachiella hugu*			KT030285	KR048861	NI
II.2. Order: Harpacticoida
Canthocamptus staphylinus*			MF077881	MF077853	NI
C. coreensis*			KT030277	KR048886	NI

To confirm the molecular taxonomy, phylogenetic analyses were conducted using obtained 28S rRNA and COI mtDNA of collected symbiotic crustaceans (7 sequences of two genes and 17 available Genbank sequences (Tab. 2). Phylogenetic trees were constructed using Bayesian inference (BI) approaches. Prior to BI analysis, best-fit models of nucleotide substitution were selected by the Akaike Information Criterion as implemented by MrModeltest 2.2 (Nylander, 2004). The best selected models were GTR+G for 28S rRNA and GTR+I+G for COI data sets, respectively. BI analyses were conducted in MrBayes 3.2.6 (Huelsenbeck and Ronquist, 2001) under the selected best-fit models and parameters. Four chains were used, and the analysis was run for 1 million generations, with the sampling frequency of 100. Each analysis was repeated twice to check for similarity of the likelihood plateau. Additionally, parameter values were evaluated for convergence throughout the run by using the ‘‘sump’’ command in MrBayes and by examining results in Tracer 1.3 (Drummond et al., 2005). Plots from Tracer were used to determine the appropriate number of trees to be discarded in the ‘‘burn in’’ and a final 50 % majority-rule consensus tree was constructed from the remaining trees.

RESULTS

Crustacean symbiont diversity and infestation status

In total, seven symbiotic crustaceans were found, with the most common symbiont being the pedunculate barnacle Octolasmis angulataAurivillius, 1894 (Prevalence 75.57 %, Mean intensity 57.26 ± 93.06). Its two congeners (O. neptuni and Octolasmis warwickiGray, 1825), and the closely related species Dianajonesia tridens Aurivillius, 1894 (WoRMS, 2020; Young, 2001) were found to have moderate abundance (> 10 %). The external morphology of these four species (Fig. 1) was consistent with previously published descriptions (Jeffries et al., 2005).

Octolasmis spp. showed the greatest variety of preferred infection sites. Octolasmis warwicki infected most of the body parts of the crabs, except for the gills, while its two congeners (O. angulata and O. neptuni) do not appear on the carapace. Dianajonesia tridens was found on most body parts, but absent from the carapace (Tab. 2, Fig. 1).

Figure 1.
Line drawings and photo images for description and distribution of infected sites of symbiotic crustaceans on P. pelagicus. Octolasmis angulata (A) and infected sites (gill lamella (above) and gill chamber (inset) (B); Octolasmis neptuni (C), on the maxilliped (D); Octolasmis warwicki (E), and on the eye (F, left), walking leg (F, right), carapace (G); Dianajonesia tridens (H), and on the coxa of cheliped (J), gill (I). Arrows indicate the symbionts. 1: Capitulum, 2: Peduncle, 3: Carina; 4: Scutum; 5: Turgum. Scale bar = 1mm.

Taxonomic records

Chelonibia testudinaria (Linnaeus, 1758)

Host and location: Portunus pelagicus, Ha Long and Kien Giang, new record for Vietnam.

Material examined and measured. Ten live specimens, including 4 specimens from a female crab (87 mm CW, 25 August 2016, locality Ha Long, collector T. Q. Sang) and 6 from a male crab (100 mm CW, 14 May 2016, locality Kien Giang, same collector).

Morphological description (Fig. 2A-D). Shell conical or evenly rounded, heavy, flat and smooth with a diameter of 7.09 ± 4.75 (1 - 15) mm (n = 10), with 6 calcium plates (1 carina, 1 rostrum, and 4 lateral), solidly joined with each other forming a hard shell surrounding the body. The orifice opening 3.33 ± 1.98 (0.5 - 6) mm long (n = 10), partially covered by 2 tergum and 2 triangular patella (Fig. 2A). Dwarf males found settled on the plates and are distributed randomly (Fig. 2B).

Infestation status. Chelonibia testudinaria is recorded as a moderately abundant species (Prevalence 14.82 %, mean intensity 9.32 ± 9.96 (Tab. 1)) and it occupied the outer surface (carapace and limbs), and inside of the sternum (Fig. 2C, D).

Figure 2.
Line drawing (A) and photo image (B) of a large individual of Chelonibia testudinaria on Portunus pelagicus. Note: Arrows indicate dwarf male distributed to the radii. Infestation sites on the host: carapace and limbs (C), and inside of the sternum (arrow) (D). 1: Carina; 2: Rostrum; 3: Lateral; 4: Orifice Opening; 5: Tergum; 6 Scutum; Scale bar = 2 mm (for A and B only).

Remarks: Chelonibia testudinaria is well-known as a successful generalist epibiotic barnacle. It is found on a wide range of marine hosts. Three species of ChelonibiaLeach, 1817 have been described: C. testudinaria on sea turtles (Rawson et al., 2003; Zardus and Hadfield, 2004; Cheang et al., 2013), Chelonibia manatiGruvel, 1903 on Sirenians, and Chelonibia patula (Ranzani, 1818) on crustaceans, e.g. blue crab (Frazier and Margaritoulis, 1990; Bakır et al., 2010; Udoh and Otoh, 2017). Based on genetic characteristics, these three species were later identified as morphotypes of the same species and synonymized under C. testudinaria (see Cheang et al., 2013; Zardus et al., 2014). Geographically, specific clades were also detected for C. testudinaria throughout its distribution range such as in the Atlantic, Indian - West Pacific, and Eastern Pacific Oceans (Rawson et al., 2003; Zardus et al., 2014). Chelonibia testudinaria was previously recorded on Portunus pelagicus (see Pasternak et al., 2002; Bakır et al., 2010; Babu et al., 2012; Sami, 2018); this, however, is the first record in Vietnam.

Semibalanus sp.

Host and Location. Portunus pelagicus, at Khanh Hoa, Vietnam, new record for the genus to both host and location.

Material examined and measured. All specimens (n = 4) from a female crab (141 mm CW, 17 Mar 2016, locality Nha Trang Bay, collector L. T. K. Oanh).

Morphological description (Fig. 3A, (B). The shell is ovoid, diameter of 3.35 ± 1.04 (2 - 4.5) mm (n = 4), with 6 plates (1 carina, 1 rostrum, 2 carinolateral and 2 lateral) truncated pyramidal, grey-white, thin, fragile, and rather smooth surface. Parietes tubiferous with a single row of major tubes. Carina trapezoidal-shaped; lateral borders overlap and do not close up with the carinolateral plates. Rostrum wide, curved trapezoidal-shaped, borders overlap the border of the laterals. Carinolateral narrow with border overlapped by carina and laterals. Orifice wide, diamond-shaped; operculum cover, diamond-shape; scutum wide, triangular with horizontal striations parallel with the basal edge and inconspicuous adductor ridge; tergum narrow and beaked with a narrow and long spur (Fig. 3A). The basis is membranous and very closely cemented to the crab carapace. The tissue inside is white (Fig. 3B).

Figure 3.
Line drawing (A) and photo image (B) of Semibalanus sp. individuals (arrows) attached to the carapace of Portunus pelagicus. Note: two unidentified gastropod snails in image. 1: Carina; 2: Carinolateral; 3: Lateral; 4: Rostrum, 5: Scutum;6: Tergum. Scale bar = 1mm for A, and 0.5 mm for B.

Infestation status. Semibalanus sp. occurred with low prevalence (0.21 %), and was only found on the carapace of one individual host crab.

Remarks. SemibalanusPilsbry, 1916 is the genus of acorn barnacle most abundant on tropical intertidal zones. Four species are currently recorded for this genus. The two common ones (Semibalanus balanoides (Linnaeus, 1767), and Semibalanus cariosus (Pallas, 1788)) are found mostly attached to rock (Takeda et al., 1998; Brousseau and Goldberg, 2007; Gyory, 2011), and rarely on crabs (McDermott, 2007); Semibalanus madrasensis (Daniel, 1958) was found on a fishing craft (Daniel, 1958), and Semibalanus sinnurensis (Daniel, 1962) on a mollusc shell (Daniel, 1962). This Semibalanus specimen shared some similar characters with S. balanoides (parallel grooves on the opening surface), and S. sinnurensis (parietes provided with minute calcareous projections), however, the observed external morphological characters (of only two individuals), and the DNA sequences are not enough for species identification. Moreover, the occurrence of Semibalanus sp. on Portunus pelagicus is assumed to be incidental.

Choniosphaera indica Gnanamuthu, 1954

Host and Location. Portunus pelagicus in Khanh Hoa, Vietnam.

Material examined and measured. All specimens (n = 5) from an ovigerous female crab (125 mm CW, 17 Mar 2016, locality Nha Trang Bay, collector L. T. K. Oanh).

Morphological description (Fig. 4A-C). The adult female is seed-like and ellipsoid in outline, size 800 - 1200 µm (1014 ± 67.04) ( 400 - 700 µm (552 ± 119.57) (n = 5), and the posterior part projects with the caudal styles. The abdomen is fused with the cephalothorax, and the esophagus occupies most of the abdominal cavity. Choniosphaera indica body is yellow or light pink with two black eyes visible on the head. The cup-like mouth tube is surrounded by the maxillae and the maxillipeds. The mouth is at the bottom of the cup and has a membranous lip (Fig. 4A1). The maxilliped is a long appendage of four unequal segments with the second one longer than the others; it has two short recurved spines and a long, comb-shaped spine on its fourth segment. Two antennas; the first antennae has five articles. The first article bears a spine, the fourth has three spines, and the terminal article carries three spines (Fig. 4A2). The second antennae are five-segmented. The third article has a spine, and the terminal article carries two long slender spines (Fig. 4A3). The tail forms two branches, assisting attachment to the crab egg (Fig. 4A, B).

Figure 4.
Line drawings (A1-4) and photo images (B, C) of Choniosphaera indica on Portunus pelagicus. External morphology (A1), first antenna (A2), second antenna (A3), and the tail (A4). C. indica eating the egg (B) and inside the egg mass (C). 1: Eyes; 2: Esophagus; 3: First antennae; 4: Second antennae; 5: Mouth tube; 6: Maxilliped; 7: Caudal portion. Scale bar = 100 mm.

Infestation status: Choniosphaera indica occurred with low prevalence (0.29 %), and moderate intensity (20.90±4.97), and was found strictly on the egg mass of the female crab host (Tab. 2, Fig. 4C), no larvae of this species were found.

Remarks. Choniosphaera spp. are specific egg eating parasites on decapod crabs. Three species have so far been recorded (Costello et al., 2001; Connolly, 2010), and among them C. indica is the most common. Choniosphaera indica was previously reported on Portunus pelagicus (Shields, 1992; Vo et al., 2013), and considered as a significant contributor to mortality of the crab host, and affecting the host population (Shields and Wood, 1993). Despite being one of the parasites capable of affecting the portunid host species population, no gene sequence of this species is available on GenBank for reference. The present study provides descriptions and images of this species, and also registers two gene sequences (28S rRNA and COI mtDNA) into GenBank.

Molecular taxonomy

In total, seven sequences were generated from each gene region of seven species of symbiotic crustacean on P. pelagicus distributing along the Vietnamese coastline. The aligned data contained unambiguous 598 bp and 921 bp of COI mtDNA and 28S rRNA genes, respectively. Compared to GenBank sequences (28S rRNA and COI mtDNA), two species (O. warwicki and C. testudinaria) showed a 100 % (EU082328 and KC138501) and 99.7 - 99.8 % (AB723914 and AY174338) identity, respectively. As for O. angulata, there is no 28S rRNA reference sequence on Genbank, however, the current COI mtDNA sequence shows 100 % matching. The remaining four species have no comparable sequences available in GenBank, therefore, comparisons were conducted with species of the same genus, and/or different genera with high proportion of similarity in a blast search (as is the case with C. indica) (see ^Appendix, Tab. A1). Conflicting results were obtained for the two genes for Semibalanus sp., with the COI sequence showing a 100 % identity match to S. cariosus (MH753555 and KM611728), but the 28S rRNA gene showing a match of only 93.4 % between the two species (MH727740 and AY520593).

Phylograms from obtained sequence data sets (28S rRNA and COI mtDNA) provided broader view of molecular taxonomy of studied symbiotic crustaceans. The BI approach applied to both data sets produced almost similar tree topologies, except the unidentified position of C. indica (Copepoda, Siphonostomatoida) from the COI tree, while it is clustered with Siphonostomatoid species (AsterocheresBoeck, 1859 and Parabrachiella C.B. Wilson, 1915) on the 28S rRNA phylogram (Fig. 5).

Figure 5.
Phylograms of Bayesian Interference trees of the symbiotic crustacean species using 28S rRNA (A) and COI mtDNA (B) data sets. The values at the tree nodes indicate Bayesian posterior probabilities. Scale bars show number of substitutions per site in the alignment.

In the 28S rRNA topology, three OctolasmisGray, 1825 species (O. angulata, O. warwicki, and Octolasmis cor (Aurivillius, 1892)) were clustered together, as a sister clade to O. neptuni + Octolasmis sp., and Octolasmis unguisiformisKobayashi and Kato, 2003 + D. tridens (Fig. 5A). Minor differences were observed in the COI tree, such as O. angulata, O. warwicki, and O. neptuni grouped as sister species, and O. cor and O. unguisiformis forming a clade (Fig. 5B). In both cases, D. tridens was grouped within the Octolasmis species, either as a sister clade to O. unguisiformis (28S rRNA) or maintained as separate clade (COI mtDNA).

The sequence differences between D. tridens and Octolasmis species ranged from 6.9 % (O. neptuni) to 9.7 % (O. unguisiformis) for 28S rRNA, and 15.7 % (O. warwicki) to 21.3 % (O. unguisiformis) for COI mtDNA. The differences between the Octolasmis species ranged from 0.7 % (O. neptuni and Octolasmis sp.) to 12.2 % (O. unguisiformis and O. warwicki), and from 14.5 % (O. angulata and O. warwicki) to 20.6 % (O. neptuni and O. unguisiformis) for 28S rRNA and COI, respectively (see ^Appendix, Tab. A2). Dianajonesia tridens was previously placed in the genus Octolamis and later moved to Dianajonesia, represented by nine species (Koçak and Kemal, 2008).

Moderate differences were also seen between the COI and 28S rRNA trees in the relative position of Semibalanus sp., which was either identical to S. cariosus (COI phylogram) or sister clade to other Semibalanus species (28S rRNA phylogram). The sequence differences were 7.6 % and 7.9 % to S. cariosus, and S. balanoides, respectively (see ^Appendix, Tab. A2). The current C. testudinaria specimen clustered in the same clade with previous C. testudinaria and conspecifics C. patula, and C. manati. They all formed a clade with Chelonibia caretta (Spengler, 1790).

DISCUSSION

Seven symbiotic crustaceans (belonging to 2 suborders, 3 families, and 5 genera) were detected on P. pelagicus in Vietnam. Of these, four species (Semibalanus sp., C. testudinaria, D. tridens, and O. neptuni) are recorded in Vietnam for the first time on P. pelagicus. Despite the intensive sampling (over 400 individual crabs), the rhizocephalan, SacculinaThompson, 1836, was not found. This is similar to the previous reports by Vo et al. (2013) on the same crab species, and Le et al. (2018b) on Portunus sanguinolentus (Herbst, 1783).

Octolasmis spp. and the genus Dianajonesia have been documented in previous studies on various host crustaceans (Shields and Overstreet, 2003; Dvoetsky and Dvoretsky, 2010; Dvoretsky, 2012; Ekanem et al., 2013) including swimmer crabs (Weng, 1987; Shield, 1992; Jeffries et al., 2005). These species were found to mainly colonize the outer surface of the host, such as the carapace, gills and gill chamber. The high density of symbionts is thought to affect the respiration and movement of hosts, exposing them to predators (Gaddes and Sumpton, 2004; Yusa et al., 2012). Although in the current study, Octolasmis species were found on almost every part of P. pelagicus body surface (Tab. 2), definite niche(s) were observed among the different species. The specific distribution may be related to the external body structure of each species. For example, O. angulata and O. neptuni have capitular plate coverage of 10 % and 16.4 %, with 3 and 5 reduced capitular plates, respectively, are found primarily on the gills (Fig. 1A, C), while O. warwicki and D. tridens, which are covered by 5 robust capitular plates (coverage 43 % and 71 %, respectively) are mainly distributed on the carapace. These findings are concordant with previous studies in terms of the number of Octolasmis species detected on swimming crabs (4 species), in the specific infestation sites, and in the structure and percent coverage of the capitular plates/capitulum (Jeffries et al., 2005).

The Semibalanus species was morphologically similar to S. balanoides; however, the molecular analysis shows it as a different taxon. We could not identify it, or describe it as a new species, due to the conflicting results from 28S rRNA and COI mtDNA sequences and limited taxonomic characters. We also found C. testudinaria, which has a well-known global distribution, and a wide variety of substrate-hosts species. Intraspecific morphological variation has previously been considered to be due to host substrate habitat differences (Rawson et al., 2003; Torres-Pratts et al., 2009; Cheang et al., 2013), and in fact, the three species of this genus reported here (C. testudinaria, C. patula, and C. manati), usually from different host taxa (marine turtles, decapod crustaceans, snakes, and Sirenia), have been combined by some (Zardus et al., 2014).

The current study provides new insights on the host and symbiont associations of swimming crabs in Vietnam and further studies could focus on the ecology of these ectosymbiont species.

ACKNOWLEDGMENTS

This project was funded by Norwegian Agency for Development Cooperation (NORAD) through the Norhed Project QZA-0485 SRV-13/0010 entitled “Incorporating Climate Change into Ecosystem Approaches to Fisheries and Aquaculture Management in Sri Lanka and Vietnam”. We thank the team members of Biodiversity and Conservation, Institute for Biotechnology and Environment, Nha Trang University for sampling support.

REFERENCES

• Abelló, P. and Corbera, J. 1996. Epibiont bryozoan (Bryozoa, Ctenostomatida) of the crab Goneplax rhomboides (Brachyura, Goneplacidae) off the Ebro delta (western Mediterranean). Miscel-lània Zoològica, 19: 43-52.
• Aurivillius, C.W.S. 1892. Neue Cirripeden aus dem Atlantischen, Indischen, und Stillen Ocean. Öfversigt af Kongliga Vetenskaps-Akademiens Förhandlingar, 3: 123-134. [In German].
• Aurivillius, C.W.S. 1894. Studien über Cirripeden. Kungliga Svenska Vetenskaps-Akademiens Handlingar, 26: 1-107. [In German].
• Babu, M.Y.; Durgekar, R.; Devi, V.J.; Ramakritinan, C.M. and Kumaraguru, A.K. 2012. Influence of cirripede barnacles Chelonibia patula (Ranzani) on commercial crabs from Gulf of Mannar and Palk bay coastal waters. Research in Environment and Life Sciences, 5: 109-116.
• Bakır, K.; Özcan, T. and Katağan, T. 2010. On the occurrence of Chelonibia patula (Cirripedia) on the coasts of Turkey. Marine Biodiversity Records, 3: 2-4.
• Boeck, A. 1859. Tvende nye parasitiske Krebsdyr, Artotrogus orbicularis og Asterocheres liljeborgii Forhandlinger i Videnskabs-Selskabet i Christiania, Aar, 2: 171-182. [In Denmark].
• Boeck, A. 1860. Tvende nye parasitiske Krebsdyr, Artotrogus orbicularis og Asterocheres Lilljeborgii. Forhandlinger i Videnskabs-Selskabet i Christiana, Aar, 2: 171-182. [In Denmark]
• Brousseau, D.J. and Goldberg, R. 2007. Effect of predation by the invasive crab Hemigrapsus sanguineus on recruiting barnacles Semibalanus balanoides in western Long Island Sound, USA. Marine Ecology Progress Series, 339: 221-228.
• Cheang, C.C.; Tsang, L.M.; Chu, K.H.; Cheng, I.J. and Chan, B.K.K. 2013. Host-Specific phenotypic plasticity of the Turtle barnacle Chelonibia testudinaria: A widespread generalist rather than a specialist. PLoS One, 8(3): e57592.
• Connolly, A. 2010. A new copepod parasite Choniosphaera cancrorum, gen. et sp. n., representing a new genus, and its larval development. Proceedings of the Zoological Society of London, 99: 415-427.
• Costello, M.; Emblow, C.S. and White, R. 2001. European register of marine species. A check-list of the marine species in Europe and a bibliography of guides to their identification. Patrimoines naturels, 50: 1-463.
• Daniel, A. 1958. A new barnacle Balanus (Semibalanus) madrasensis n. sp. from fishing craft off Madras. Journal of Natural History Series, 13: 305-308.
• Daniel, A. 1962. On a new species of Operculate barnacle (Cirripedia: Crustacea) from the Gastropod mollusc, Murex sp. from Porto Novo, Madras State. Annals and Magazine of Natural History, 13: 193-197.
• Drummond, A.J.; Rambaut, A.; Shapiro, B. and Pybus, O.G. 2005. Bayesian coalescent inference of past population dynamics from molecular sequences. Molecular Biology and Evolution, 22: 1185-1192.
• Dvoretsky, A.G. 2012. Epibionts of the great spider crab, Hyas araneus (Linnaeus, 1758), in the Barents Sea. Polar Biology, 35: 625-631.
• Dvoretsky, A.G. and Dvoretsky, V.G. 2010. Epifauna associated with an introduced crab in the Barents Sea: A 5-year study. ICES Journal of Marine Science, 67: 204-214.
• Ekanem, A.P.; Eyo, V.O.; Ekpo, I.E.; Bassey, B.O. and Ekanem, A.P. 2013. Parasites of Blue Crab (Callinectes amnicola) in the Cross River Estuary, Nigeria. International Journal of Fisheries and Aquatic Studies, 1: 18-21.
• Filipiak, A.; Zaj, K.; Kübler, D. and Kramarz, P. 2016. Coevolution of cophylogeny associations and methods for studying their cophylogeny. Invertebrate Survival Journal, 13: 56-65.
• Folmer, O.; Black, M.; Hoeh, W.; Lutz, R. and Vrijenhoek, R. 1994. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology, 3: 294-299.
• Frazier, J.G. and Margaritoulis, D. 1990. Notes and News: The occurrence of the barnacle, Chelonibia patula (Ranzani, 1818), on an Inanimate Substratum (Cirripedia, Thoracica). Crustaceana, 59: 213-218.
• Gaddes, S.W. and Sumpton, W.D. 2004. Distribution of barnacle epizoites of the crab Portunus pelagicus in the Moreton Bay region, eastern Australia. Marine and Freshwater Research, 55: 241-248.
• Galil, B.S. and Innocenti, G. 1999. Notes on the population structure of the portunid crab Charybdis longicolis Leene, parasitized by the rhizocephalan Heterosaccus dolffusi Boschma, off the Mediterranean coast of Israel. Bulletin of Marine Science, 64: 451-463.
• Gnanamuthu, C.P, 1954. Choniosphaera indica, a copepod parasitic on the crab Neptunus sp. Parasitology, 44: 371-378.
• Gray, J.E. 1825. A synopsis of the genera of cirripedes arranged in natural families, with a description of some new species. Annals of Philosophy, 10: 97-107.
• Gruvel, J.A. 1903. Révision des Cirripèdes appartenant à la collection du Muséum national d'Histoire Naturelle (Operculés). I. Partie systématique. Nouvelles Archives du Muséum national d'Histoire naturelle, 4, 5: 95-170. [In French].
• Gyory, J. 2011. Larval ecology and synchronous reproduction of two crustacean species: Semibalanus balanoides in New England, USA and Gecarcinus quadratus in Veraguas, Panama. Cambridge, Massachusetts Institute of Technology and Woods Hole Oceanographic Institution, Ph.D. Thesis, 146p.
• Hall, T.A. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series, 41: 95-98.
• Herbst, J.F.W. 1783. Kritisches Verzeichniß meiner Insektensammlung. Archiv der Insectengeschichte, Zürich, 4: 1-72. [In German].
• Hudson, D.A. and Lester, R.J.G. 1994. Parasites and symbionts of wild mud crabs Scylla serrata (Forskal) of potential significance in aquaculture. Aquaculture, 120: 183-199.
• Huelsenbeck, J.P. and Ronquist, F., 2001. MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics, 17: 754-755.
• Isaeva, V. V.; Dolganov, S.M. and Shukalyuk, A.I. 2005. Rhizocephalan barnacles - Parasites of commercially important crabs and other decapods. Russian Journal of Marine Biology, 31: 215-220.
• Jeffries, W.B. and Voris, H.K. 1996. A subject-indexed bibliography of the symbiotic barnacles of the genus Octolasmis Gray, 1825 (Crustacea: Cirripedia: Poecilasmatidae). The Raffles Bulletin of Zoology, 44: 575-592.
• Jeffries, W.B.; Voris, H.K.; Naiyanetr, P. and Panha, S. 2005. Pedunculate barnacles of the symbiotic genus Octolasmis (Cirripedia: Thoracica: Poecilasmatidae) from the Northern Gulf of Thailand. The Natural History Journal of Chulalongkorn University, 5: 9-13.
• Kearse, M.; Moir, R.; Wilson, A.; Stones-Havas, S.; Cheung, M.; Sturrock, S.; Buxton, S.; Cooper, A.; Markowitz, S.; Duran, C.; Thierer, T.; Ashton, B.; Meintjes, P. and Drummond, A. 2012. Geneious Basic: An integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics, 28: 1647-1649.
• Klinbunga, S.; Khetpu, Æ.K.; Khamnamtong, B. and Menasveta, Æ.P. 2007. Genetic Heterogeneity of the Blue Swimming Crab (Portunus pelagicus) in Thailand Determined by AFLP analysis. Biochemical Genetics, 45: 725-736.
• Kobayashi, C. and Kato, M. 2003. Sex-biased ectosymbiosis of a unique cirripede, Octolasmis unguisiformis sp. nov., that resembles the chelipeds of its host crab, Macrophthalmus milloti Journal of the Marine Biological Association of the United Kingdom, 83: 925-930.
• Koçak, A.Ö. and Kemal, M. 2008. Notes on the nomenclature of some genus group names in Arthropoda. Miscellaneous Papers,Centre for Entomological Studies, Ankara, 138: 2.
• Kunsook, C.; Gajaseni, N. and Paphavasit, N. 2014a. A stock assessment of the blue swimming crab Portunus pelagicus (Linnaeus, 1758) for sustainable management in Kung Krabaen Bay, Gulf of Thailand. Tropical Life Sciences Research, 25: 41-59.
• Kunsook, C.; Gajaseni, N. and Paphavasit, N. 2014b. The feeding ecology of the blue swimming crab, Portunus pelagicus (Linnaeus, 1758), at Kung Krabaen bay, Chanthaburi Province, Thailand. Tropical Life Sciences Research, 25: 13-27.
• Kwiatkowski, M.; Engelstädter, J. and Vorburger, C. 2012. On Genetic Specificity in Symbiont-Mediated Host-Parasite Coevolution. PLOS Computational Biology, 8(8): e1002633.
• Leach, A. 1817. The Sessile Barnacles. Journal de Physique, 85: 1-69
• Le, O.T.; Dang, B. and Tran, S. 2018a. Infestation status of epizoic barnacle Octolasmis warwicki on blue swimming crab Portunus pelagicus at Khanh Hoa and Phu Yen province. Journal of Tropical Science and Technology, 15: 34-41. [In Vietnamese].
• Le, O.T.; Vo, T. and Nguyen, T.T. 2018b. Some ectoparasites on three-spot swimming crab (Portunus sanguinolentus, Herbst 1783) in Khanh Hoa Province. Journal of Tropical Science and Technology, 17: 28-38. [In Vietnamese].
• Linnaeus, C. 1758. Systema Naturae per Regna Tria Naturae: Secundum Classes, Ordines, Genera, Species cum Characteribus, Differentiis, Synonymis, Locis. Zug, Switzerland: Inter Documentation Centre, 10: 1-824.
• Linnaeus, C. 1767. Systema naturae per regna tria naturae, secundum classes, ordines, genera, species, cum characteribus, differentiis, synonymis, locis. Stockholm: Laurentius Salvius, 736p.
• MacDonald, J. 1869. On an apparently new genus of minute parasitic cirriped, between Lepas and Dichelapsis Journal of Zoology, 37: 440-444.
• Machado, G.B.O.; Sanches, F.H.C.; Fortuna, M.D. and Costa, T.M. 2013. Epibiosis in decapod crustaceans by stalked barnacle Octolasmis lowei (Cirripedia: Poecilasmatidae). Zoologia, 30: 307-311.
• Margolis, L.; Esch, G.W.; Holmes, J.C.; Kuris, A.M. and Schad, G.A. 1982. The use of ecological terms in parasitology (Report of an Ad Hoc Committee of the American Society of Parasitologists). The Journal of Parasitology, 68: 131-133.
• McDermott, J.J 2007. Ectosymbionts of the non-indigenous Asian shore crab, Hemigrapsus sanguineus (Decapoda: Varunidae), in the western north Atlantic, and a search for its parasites. Journal of Natural History, 41: 2379-2396.
• Miers, E.J. 1876. Catalogue of the Stalk-and Sessile-Eyed Crustacea of New Zealand. Colonial Museum and Geological Survey Department, London, 184p.
• Mizrahi, I.; Achituv, Y.; Katcoff, D.J. and Perl Treves, R. 1998. Phylogenetic position of Ibla (Cirripedia: Thoracica) based on 18S rDNA sequence analysis. Journal of Crustacean Biology, 18: 363-368.
• Nylander, J.A.A. 2004. MrModeltest v2. Program distributed by the author. Evolutionary Biology Centre, Uppsala University.
• Olson, P.D.; Cribb, T.H.; Tkach, V. V.; Bray, R.A. and Littlewood, D.T.J. 2003. Phylogeny and classification of the Digenea (Platyhelminthes: Trematoda). International Journal for Parasitology, 33: 733-755.
• Pallas, P.S. 1788. Marina varia nova et rariora. Nova Acta Academiae Scientiarum Imperialis Petropolitanae, 2: 229-249.
• Pasternak, Z.; Abelson, A. and Achituv, Y. 2002. Orientation of Chelonibia patula (Crustacea: Cirripedia) on the carapace of its crab host is determined by the feeding mechanism of the adult barnacles. Journal of the Marine Biological Association of the United Kingdom, 82: 583-588.
• Pilsbry, H.A. 1916. The sessile barnacles (Cirripedia) contained in the collections of the U.S. National Museum; including a monograph of the American species. Bulletin of the United States National Museum, 93: 1-366.
• Ranzani, C. 1818. Osservazioni su i Balanidi. Opuscoli Scientifici, Bologna, 1: 269-276.
• Rawson, P.D.; Macnamee, R.; Frick, M.G. and Williams, K.L. 2003. Phylogeography of the coronulid barnacle, Chelonibia testudinaria, from loggerhead sea turtles, Caretta caretta Molecular Ecology, 12: 2697-2706.
• Rózsa, L.; Reiczigel, J. and Majoros, G. 2000. Quantifying parasites in samples of hosts. Journal of Parasitology, 86: 228-232.
• Sami, K. 2018. The first record of the barnacle Chelonibia patula (Ranzani, 1818), hosted by the swimming crab Portunus segnis (Forskål, 1775), in the Gulf of Gabès. Bulletin de l'Institut National des Sciences et Technologies de la Mer de Salammbô, Number Special: 89-91.
• Shields, J.D. 1992. Parasites and symbionts of the crab Portunus pelagicus from Moreton Bay, eastern Australia. Journal of Crustacean Biology, 12: 94-100.
• Shields, J. and Overstreet, R.M. 2003. The Blue Crab: Diseases, Parasites, and other Symbionts. Faculty Publications from the Harold W. Manter Laboratory of Parasitology. Lincoln, University of Nebraska, 426p.
• Shields, J.D. and Wood, F.E.I. 1993. Impact of parasites on the reproduction and fecundity of the blue sand crab Portunus pelagicus from Moreton Bay, Australia. Marine Ecology Progress Series, 92: 159-170.
• Spengler, L. 1790. Beskrivelse og Oplysingover den hidindtil lidet udarbeide Slaegtaf mangeskallede Konchylier, som Linnaeus har daldet Lepas, med tilfoiede nye ogubeskrevne Aeter (Om. Conchylie-SlaegtenLepas). Skrifter af Naturhistorie Selskabet, 1: 158-212.
• Takakura, U. 1910. Kisei himomushi no ichi Shinshu (A new species of parasitic nemertean). Zoological Magazine, Tokyo, 22: 111 -116. [In Japanese].
• Takeda, S.; Shimokawa, Y. and Murakami, O. 1998. Daily activity of the Barnacle, Semibalanus cariosus (Pallas). Crustaceana, 71: 299-311.
• Thompson, J.V. 1836. Natural history and metamorphosis of an anomalous crustaceous parasite of Carcinus maenas, the Sacculina carcini Entomological Magazine, 3: 452-456.
• Torres-Pratts, H.; Schärer, M.T. and Schizas, N. V. 2009. Genetic diversity of Chelonibia caretta, commensal barnacles of the endangered hawksbill sea turtle Eretmochelys imbricata from the Caribbean (Puerto Rico). Journal of the Marine Biological Association of the United Kingdom, 89: 719-725.
• Udoh, J.P. and Otoh, A.J. 2017. Distribution and size of barnacle Chelonibia patula fouling blue brab Callinectes amnicola in southeast Nigeria. Croatian Journal of Fisheries, 74: 93-102.
• VASEP, 2021. Report on Vietnam seafood exports in Quarterly III/2021. Availabe at Availabe at https://www.seafood.vasep.com.vn/reports/quarterly-report-on-vietnam-seafood-exports/ reports-on-vietnam-seafood-export/report-on-vietnam-seafood-exports-in-q-iii-2021-22071.html Accessed on 27 May 2022
  » https://www.seafood.vasep.com.vn/reports/quarterly-report-on-vietnam-seafood-exports/ reports-on-vietnam-seafood-export/report-on-vietnam-seafood-exports-in-q-iii-2021-22071.html
• Vo, T.D.; Bristow, G.A.; Pham, N.H. and Nguyen, T.H.T. 2013. Some parasites found from swimming crab (Portunus pelagicus Linnaeus, 1766) caught in Khanh Hoa marine water. Journal of Fisheries Science and Technology, Nha Trang University, 3: 11-15. [In Vietnamese].
• Vu, V.H.; Hoang, N.T.; Tran, C.V. and Nguyen, S. 2014. Stock and fishery assessment report of blue swimming crab Portunus pelagicus (Linnaeus, 1758) in Kien Giang waters, Viet Nam. Report for WWF and WASEP. Hai Phong, Research Institute for Marine Sciences, Department of Marine Fisheries Rosources Fisheries, 52p.
• Weng, H. 1987. The Parasitic barnacle, Sacculina granifera Boschma, affecting the commercial sand crab Portunus pelagicus (L.), in populations from two different environments in Queensland. Journal of Fish Diseases, 10: 221-227.
• Wilson, C.B. 1915. North American parasitic copepods belonging to the Lernaeopodidae, with a revision of the entire family. Proceedings of the United States National Museum, 47(2063): 565-729.
• WoRMS, 2020. Dianajonesia tridens (Aurivillius, 1894). Available at Available at http://www.marinespecies.org/aphia.php?p=taxdetails&id=1423498 Accessed on 07 July 2020.
  » http://www.marinespecies.org/aphia.php?p=taxdetails&id=1423498
• Wu, T.H. 2011. Phylogeny and Population Genetics of Acorn Barnacles in Family Chthamalidae (Crustacea: Cirripedia). Hong Kong, The Chinese University of Hong Kong, Master thesis, 123p.
• Young, P.S. 2001. Deep-sea Cirripedia Thoracica (Crustacea) from the northeastern Atlantic collected by French expeditions. Zoosystema, 23: 705-756.
• Yusa, Y.; Yoshikawa, M.; Kitaura, J.; Kawane, M.; Ozaki, Y.; Yamato, S. and Høeg, J.T. 2012. Adaptive evolution of sexual systems in pedunculate barnacles. Proceedings of the Royal Society B: Biological Sciences, 279: 959-966.
• Zardus, J.D. and Hadfield, M.G. 2004. Larval development and complemental males in Chelonibia testudinaria, a barnacle commensal with sea turtles. Journal of Crustacean Biology, 24: 409-421.
• Zardus, J.D.; Lake, D.T.; Frick, M.G. and Rawson, P.D. 2014. Deconstructing an assemblage of “turtle” barnacles: Species assignments and fickle fidelity in Chelonibia Marine Biology, 161: 45-59.

• Zoobank:

  http://zoobank.org/urn:lsid:zoobank.org:pub: B2A670A9-01D2-492B-B8D9-61E48DBFF93C

APPENDIX

Figure A1.
Line drawing showing the external morphology of the blue swimming crab (half dorsal and cut-away views) with dissected body parts (numbered from 1-5) used for recording the symbiotic organisms.

Table A1.
Results of Blast Nucleotide search for 28S rRNA and COI mtDNA gene sequences of studied species with the sequences retrieved from Genbank. GB - GenBank; X - sequences not available

No.	Studied species	28S rRNA				COI mtDNA
		Reference species	Query cover (%)	% Identity	GB Accession no.	Query cover (%)	% Identity	GB Accession no.
1	Octolasmis angulata	Octolasmis cor	100	98.1	EU082326	X	X
		Octolasmis unguisiformis	99	91.5	LC467957	X	X
		Octolasmis angulata	X	X		100	100	KC138498
2	Octolasmis neptuni	Octolasmis sp.	100	99.2	EU082327	X	X
		Octolasmis unguisiformis	99	91	LC467957	X	X
		Octolasmis cor	X	X		100	98.9	KC138500
3	Octolasmis warwicki	Octolasmis warwicki	100	100	EU082328	100	100	KC138501
		Octolasmis cor	100	93	EU082326	X	X
4	Dianajonesia tridens	Octolasmis cor	100	93	EU082326	X	X
		Octolasmis sp.	100	93	EU082327	X	X
		Dichelaspis hawaiense	X	X		100	84.5	KF484230
5	Chelonibia testudinaria	Chelonibia patula	100	99.9	EU082295	100	99.8	JF823668
		Chelonibia manati	100	99.7	AB723917	X	X
		Chelonibia testudinaria	100	99.7	AB723914	100	99.8	AY174338
6	Semibalanus sp.	Semibalanus cariosus	100	93.4	AY520593	100	100	KM611728
		Semibalanus balanoides	100	93.4	EU370440	X	X
7	Choniosphaera indica	Asterocheres aesthetes	69	90.3	KR048857	X	X
		Asterocheres lilljeborgi	66	91	KR048868	X	X

Table A2
Percentage (%) of sequence differences (above diagonal) and identities (below diagonal) of 28S rRNA (A) and COI mtDNA (B) genes following the taxonomic families of study species. The largest different/identity values are bolded and highlighted in red.

A. 28S rRNA
I. Subclass: Thecostraca
I.I. Superorder: Thoracica, Order: Lepadiformes
Family: Poecilasmatidae
Seq->		1	2	3	4	5	6	7
1	Octolasmis angulata	ID	6,1	7,1	6,5	1,9	10,1	7,3
2	Octolasmis neptuni	93,9	ID	8,3	0,7	6,3	10,4	6,9
3	Octolasmis warwicki	92,9	91,7	ID	8,4	7,4	12,2	9,2
4	Octolasmis sp. EU082327	93,5	99,3	91,6	ID	6,6	10,7	7,3
5	Octolasmis cor EU082326	98,1	93,7	92,6	93,4	ID	11	7,3
6	Octolasmis unguisiformis LC467957	89,9	89,6	87,8	89,3	89	ID	9,7
7	Dianajonesia tridens	92,7	93,1	90,8	92,7	92,7	90,3	ID
I.2. Superorder: Thoracica, Order: Sessilia
Family: Balanidae
Seq->		1	2	3
1	Semibalanus sp.	ID	7,6	7,9
2	Semibalanus cariosus AY520593	92,4	ID	1,3
3	Semibalanus balanoides EU370440	92,1	98,7	ID
Family: Chelonibiidae
Seq->		1	2	3	4	5	6	7
1	Chelonibia testudinaria	ID	0,3	0,4	2,7	0,2	4,4	6,1
2	Chelonibia manati AB723917	99,7	ID	0,4	2,7	0,2	4,7	6,4
3	Chelonibia testudinaria AB723914	99,6	99,6	ID	2,3	0,3	4,4	6,1
4	Chelonibia testudinaria KM217527	97,3	97,3	97,7	ID	2,5	5,7	4
5	Chelonibia patula EU082295	99,8	99,8	99,7	97,5	ID	4,6	6,3
6	Chelonibia caretta AB723915	95,6	95,3	95,6	94,3	95,4	ID	2,4
7	Chelonibia caretta KM217526	93,9	93,6	93,9	96	93,7	97,6	ID
II. Subclass: Copepoda
Superorder: Podoplea
Order: Siphonostomatoida
Seq->		1	2	3	4	5
1	Choniosphaera indica	ID	24,9	27,3	29,7	30,3
2	Asterocheres lilljeborgi KR048868	75,1	ID	18,6	20,1	21,8
3	Parabrachiella hugu KR048861	72,7	81,4	ID	24,6	25,8
Order: Harpacticoida
4	Canthocamptus staphylinus MF077853	70,3	79,9	75,4	ID	10,2
5	Canthocamptus coreensis KR048886	69,7	78,2	74,2	89,8	ID
B. COI mtDNA
I. Subclass: Thecostraca
I.I. Superorder: Thoracica, Order: Lepadiformes
Family: Poecilasmatidae
Seq->		1	2	3	4	5	6
1	Octolasmis angulata	ID	16	14,5	17,6	18,7	17,8
2	Octolasmis neptuni	84	ID	14,7	19,7	20,6	18,1
3	Octolasmis warwicki	85,5	85,3	ID	16,9	17,8	15,7
4	Octolasmis cor KC138499	82,4	80,3	83,1	ID	19,4	18,8
5	Octolasmis unguisiformis LC467960	81,3	79,4	82,2	80,6	ID	21,3
6	Dianajonesia tridens	82,2	81,9	84,3	81,2	78,7	ID
I.2. Superorder: Thoracica, Order: Sessilia
Family: Balanidae
Seq->		1	2	3
1	Semibalanus sp.	ID	0	14,7
2	Semibalanus cariosus KM611728	100	ID	14,7
Family: Chelonibiidae
Seq->		1	2	3	4	5	6	7
1	Chelonibia testudinaria	ID	10,3	1,1	1,3	0,7	17,6	17,4
2	Chelonibia manati JN589813	89,7	ID	10	9,6	9,8	16,7	16,6
3	Chelonibia testudinaria KF042514	98,9	90	ID	0,9	0,4	17,1	16,9
4	Chelonibia testudinaria KF042515	98,7	90,4	99,1	ID	0,6	17,3	17,1
5	Chelonibia patula JF823674	99,3	90,2	99,6	99,4	ID	17,3	17,1
6	Chelonibia caretta KF042512	82,4	83,3	82,9	82,7	82,7	ID	0,7
7	Chelonibia caretta KF042513	82,6	83,4	83,1	82,9	82,9	99,3	ID
II. Subclass: Copepoda
Superorder: Podoplea
Order: Siphonostomatoida
Seq->		1	2	3	4	5
1	Choniosphaera indica	ID	42,2	44,5	43,3	43,3
2	Asterocheres lilljeborgi KR049050	57,8	ID	26,6	29,5	28,3
3	Parabrachiella hugu KT030285	55,5	73,4	ID	30,2	21,4
Order: Harpacticoida
4	Canthocamptus staphylinus MF077881	56,7	70,5	69,8	ID	22,6
5	Canthocamptus coreensis KT030277	56,7	71,7	78,6	77,4	ID

Publication Dates

• Publication in this collection
  16 Sept 2022
• Date of issue
  2022

History

• Received
  25 Aug 2021
• Accepted
  28 Feb 2022