Mitochondrial genome of the intertidal acorn barnacle Tetraclita serrata Darwin, 1854 (Crustacea: Sessilia): Gene order comparison and phylogenetic consideration within Sessilia

Marine Genomics 22 (2015) 63–69

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Mitochondrial genome of the intertidal acorn barnacle Tetraclita serrata Darwin, 1854 (Crustacea: Sessilia): Gene order comparison and phylogenetic consideration within Sessilia Xin Shen a,b,1, Ling Ming Tsang c,1, Ka Hou Chu b, Yair Achituv d, Benny Kwok Kan Chan e,⁎ a Jiangsu Key Laboratory of Marine Biotechnology/College of Marine Science/Co-Innovation Center of Jiangsu Marine Bio-industry Technology, Huaihai Institute of Technology, Lianyungang 222005, China b Simon F. S. Li Marine Science Laboratory, School of Life Sciences, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China c Institute of Marine Biology, National Taiwan Ocean University, Keelung 202, Taiwan d The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel e Biodiversity Research Center, Academia Sinica, Taipei 115, Taiwan

a r t i c l e

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Article history: Received 4 December 2014 Received in revised form 9 April 2015 Accepted 9 April 2015 Available online 20 April 2015 Keywords: Crustacea Sessilia Tetraclita serrata Mitochondrial genome Gene rearrangement Phylogeny

a b s t r a c t The complete mitochondrial genome of the intertidal barnacle Tetraclita serrata Darwin, 1854 (Crustacea: Maxillopoda: Sessilia) is presented. The genome is a circular molecule of 15,200 bp, which encodes 13 PCGs, 2 ribosomal RNA genes, and 22 transfer RNA genes. All non-coding regions are 591 bp in length, with the longest one speculated as the control region (389 bp), which is located between srRNA and trnK. The overall A + T content of the mitochondrial genome of T. serrata is 65.4%, which is lowest among all the eight mitochondrial genomes reported from sessile barnacles. There are variations of initiation and stop codons in the reported sessile barnacle mitochondrial genomes. Large-scale gene rearrangements are found in these genomes as compared to the pancrustacean ground pattern. ML and Bayesian analyses of all 15 complete mitochondrial genomes available from Maxillopoda lead to identical phylogenies. The phylogenetic tree based on mitochondrial PCGs shows that Argulus americanus (Branchiura) cluster with Armillifer armillatus (Pentastomida), distinct from all ten species from Cirripedia. Within the order Sessilia, Amphibalanus amphitrite (Balanidae) clusters with Striatobalanus amaryllis (Archaeobalanidae), and Nobia grandis (Pyrgomatidae). However, the two Megabalanus (Balanidae) are separated from the above grouping, resulting in non-monophyly of the family Balanidae. Moreover, the two Megabalanus have large-scale rearrangements as compared to the gene order shared by former three species. Therefore, both phylogenetic analysis using PCG sequences and gene order comparison suggest that Balanidae is not a monophyletic group. Given the limited taxa and moderate support values of the internal branches, the non-monophyly of the family Balanidae requires further verification. © 2015 Elsevier B.V. All rights reserved.

1. Introduction With a few exceptions, metazoan mitochondrial DNA are circular molecules, 13–20 kb in size, typically containing 37 genes: 13 PCGs (cox1–3, cob, nd1–6, nd4L, atp6 and atp8), 2 rRNAs (srRNA and lrRNA), and 22 tRNAs. Comparisons of complete mitochondrial genomes have Abbreviations: atp6 and 8, ATPase subunits 6 and 8; cox1–3, cytochrome c oxidase subunits I–III; PCGs, protein coding genes; cob, cytochrome b; nd1–6 and 4 L, NADH dehydrogenase subunits 1–6 and 4 L; srRNA and lrRNA, small and large subunits ribosomal RNA; tRNA, transfer RNA; L1, tRNALeu(CUN); L2, tRNALeu(UUR); S1, tRNASer(AGY); S2, tRNASer(UCN). ⁎ Corresponding author. Tel.: +886 2 27872231. E-mail address: [email protected] (B.K.K. Chan). 1 Both of these authors contributed equally to this work.

http://dx.doi.org/10.1016/j.margen.2015.04.004 1874-7787/© 2015 Elsevier B.V. All rights reserved.

been extensively used for deducing metazoan phylogeny (Krause et al., 2010; Lindqvist et al., 2010; Morin et al., 2010). In fact, complete mitochondrial genomes are more informative than single gene or gene fragments, and they can reveal some genome-level features (Shen et al., 2009). Tetraclitidae Gruvel, 1903 (Crustacea: Sessilia) contains approximately 94 extant and 114 fossil known species that are distributed among at least four subfamilies and 16 genera. Tetraclita species are common rocky intertidal acorn barnacles in the tropical and subtropical waters of the world (Chan et al., 2007a, 2007b, 2008; Shahdadi et al., 2011). Tetraclita serrata is a common intertidal barnacle in the southern African waters. Here we present the complete mitochondrial genome of T. serrata (Crustacea: Maxillopoda: Sessilia). The gene content,

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arrangement and PCGs of the mitochondrial genome were compared with those genomes available from all barnacles. Such comparison allows investigation of the phylogenetic relationships of the order Sessilia.

(Zuker, 2003). Gene map of T. serrata mitochondrial genome was drawn by OGDraw 1.2 (Lohse et al., 2013). Furthermore, codon usage in the 13 PCGs of the mitochondrial genome was estimated with DnaSP 5.10.01 (Librado and Rozas, 2009).

2. Materials and methods

2.4. Phylogenetic analysis

2.1. Sample collection and DNA extraction

Along with newly obtained mitochondrial genome sequence of T. serrata, the currently available 14 mitochondrial genomes from Maxillopoda (Table S1) were used in phylogenetic analysis, including seven genomes from Sessilia (Megabalanus volcano, Megabalanus ajax, Notochthamalus scabrosus, Tetraclita japonica, Nobia grandis, Striatobalanus amaryllis and Amphibalanus amphitrite) (Shen et al., 2014a, 2014b; Tsang et al., 2014b, 2014c; Wares, 2015), two from Pedunculata (Capitulum mitella and Pollicipes polymerus) (Lavrov et al., 2004), three from Copepoda (Calanus hyperboreus, Tigriopus californicus and Tigriopus japonicus) (Machida et al., 2002; Burton et al., 2007; Kim et al., 2013), and one each from Branchiura (Argulus americanus) and Pentastomida (Armillifer armillatus) (Lavrov et al., 2004). Nucleotide sequences of the 13 PCGs from these mitochondrial genomes were aligned using Clustal X with the default settings (Larkin et al., 2007), and were concatenated into a single alignment. The final alignment for the 15 genomes consisted of 11,829 sites. To determine the best fitting model, a nested likelihood ratio test was performed using jModelTest 2 (Darriba et al., 2012). After the evolutionary model was determined (GTR + I + G), maximum likelihood (ML) and Bayesian inference (BI) analyses were performed using PhyML 3.0 (Guindon et al., 2010) and MrBayes 3.2 (Ronquist et al., 2012), respectively. For ML analysis, 1000 bootstraps were used to estimate the nodal reliability (BPM). As for BI, the Markov Chain Monte Carlo analysis (four chains) was run for 1,000,000 generations (sampling every 1000 generations) to allow adequate time for convergence. After approximate 200,000 generations, the log-likelihood values of each sampled tree had stabilized. The first 200 trees were excluded from the analysis as “burn-in”, while the remaining 800 trees were used to estimate the Bayesian posterior probabilities (BPP).

The specimen of T. serrata was obtained from Betty Bay, South Africa (Tsang et al., 2012). The muscle tissue from the fresh specimen was immediately preserved in 95% ethanol. Total genomic DNA was extracted from adductor or abdominal muscle tissue, using the commercial QIAamp Tissue Kit (QIAGEN). 2.2. PCR and sequence determination Initially, we used degenerate primer sets to amplify the cob, cox3 and nd2 (Table 1) from the mitochondrial genome. The PCR amplifications were performed with the following cycling parameters: initial denaturation at 94 °C for 3 min, five cycles of denaturation at 92 °C for 15 sec, annealing at 30 °C for 15 sec, elongation at 68 °C for 1 min, followed by 10 cycles of denaturation at 92 °C for 15 sec, annealing at 42 °C for 15 sec, elongation at 68 °C for 1 min, and finally 30 cycles of denaturation at 92 °C for 15 sec, annealing at 50 °C for 15 sec, elongation at 68 °C for 1 min and a final extension at 68 °C for 6 min. PCR products were purified (GeneMark), cloned (pGEMT easy, Promega) and sequenced. Species specific primer sets (CC1BS-F: GAA ATG GAA GAG CAG GCT AAG ATT TTT CGT A, CC1BS-R: TCG GGT GAG GGT AGC ATT ATC AAC GGC G, CC1CS-F: CAT CGA AGC TCC GTT CTC AAT CGC CGA G, CC1CSR: CTC GGC GAT TGA GAA CGG AGC TTC GAT G, CC1DS-F: AGA ACT TCT GGA AAC CAG AAA TGG AAT GGG GC, and CC1DS-R: GCC CCA TTC CAT TTC TGG TTT CCA GAA GTT CT) were designed based on the sequences for long range PCR to amplify the complete mitochondrial genomes. AccuPrime Taq DNA polymerase (Invitrogen) was used in long range PCR with the following cycling profile: initial denaturation at 94 °C for 3 min, followed by 40 cycles of denaturation at 92 °C for 15 sec, elongation at 68 °C for 6 min and a final extension at 68 °C for 10 min. The PCR products were cloned into the pGEMT easy vector (Promega) and transformed in Escherichia coli competent cells. Plasmids were sequenced on T7, SP6 and walking primers by using the BigDye version 3.1 kit (Applied Biosystems) on an ABI 3730XL analyzer (Applied Biosystems). 2.3. Gene identification and genome analysis The sequences were aligned using SeqMan 7.1 (Lasergene). A total of 22 reads were joined to form a complete mitochondrial genome. The boundaries of 13 PCGs and two rRNAs were initially determined with DOGMA (Wyman et al., 2004), and then refined by alignment with mitochondrial genomes available from barnacles. The location and orientation of most tRNAs were identified using tRNAscan-SE 1.21 (Schattner et al., 2005) under the default mode. The remaining tRNA genes were identified by sequences alignment and RNA folding

3. Results and discussion 3.1. General features The mitochondrial genome of T. serrata is a circular molecule of 15,200 bp, which is similar to those from other sessile barnacles. It encodes 13 PCGs, 2 rRNA genes, and 22 tRNA genes. The heavy and light strands contain 24 and 13 genes, respectively (Fig. 1; Table 2). Due to the compactness of the mitochondrial genome, seven instances of gene overlaps are found in T. serrata. Two 7-bp overlaps are found in atp8/atp6 and nd4/nd4L genes, respectively. There are five other overlaps ranging from 1 to 2 bp. Non-coding regions make up 591 bp, with the longest one speculated as the control region (389 bp), which is located between srRNA and trnK (Table 2). The overall A + T content of the mitochondrial genome of T. serrata is 65.4%, which is lower than the values from all the other seven studied sessile barnacles (ranging from 66.1% to 73.1%). The entire T. serrata mitochondrial genome sequence was deposited in GenBank with accession number KJ434948. 3.2. Protein-coding genes

Table 1 Primers used to amplify the mitochondrial genes of Tetraclita serrata (Crustacea: Sessilia). Gene

Primer

Sequence (5′–3′)

cob

bar_cob_F bar_cob_R bar_C3_F bar_C3_R bar_nd2_F bar_nd2_R

TCATATTATACGWGAYGTWAAYAGAGGATG ATAAGGGTCTTCTACWGGYATMCCTCCAAT GCCCCTTCAGTNGAAATTGG ACTACATCDACRAAATGTCAATATCA GCYCCATTYCAYTTCTGRTTYCC GAAATRGADGARYARGCTAARATTTTTCG

cox3 nd2

In T. serrata mitochondrial genome, four PCGs (nd1, nd4, nd4L and nd5) are encoded on the light strand while the other nine PCGs are located on the heavy strand. In all eight sessile barnacle mitochondrial genomes, the numbers of amino acids in five of the PCGs (cob, cox2, cox3, nd3 and atp8) are identical respectively. Yet there are gene length variations in the remaining eight PCGs. Metazoan mitochondrial PCGs often use several ATN alternatives as start codons (Shen et al., 2009, 2012b). However, all 13 PCGs in T. serrata start with ATN (ATG, ATA or

X. Shen et al. / Marine Genomics 22 (2015) 63–69

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Fig. 1. Gene map of mitochondrial genome of Tetraclita serrata Darwin, 1854 (Crustacea: Sessilia). Transfer RNA genes are designated amino acid codes. Inner ring displays the GC content. Genes encoded on the heavy and light strands are shown outside and inside the circular gene map, respectively.

ATC) (Table 2). On the other hand, PCGs of the two Megabalanus mitochondrial genomes have initiation codons other than “ATN”. The nd4L gene in both genomes has the initiation codon “GTG”. Further, the cox1 gene of M. ajax and M. volcano starts with “CTT” and “TTG”, respectively. The nd4L gene of A. amphitrite also uses “GTG” as the start codon. Three PCGs (cox3, nd3 and nd4) in T. serrata end with incomplete stop codons (T-), and the remaining ones have complete stop codons (TAA or TAG). On the other hand, all complete stop codons in the N. grandis mitochondrial genome are TAA. Therefore, there are initiation and stop codon variations in the eight sessile barnacle mitochondrial genomes. The pattern of codon usage in the T. serrata mitochondrial genome was also studied. There are 3689 codons in all the 13 PCGs (excluding the incomplete termination). The most frequently used amino acids were Leu (15.53%), followed by Phe (9.76%), Ser (9.57%) and Ile (8.24%) (Table 3). A common feature in most metazoan genomes is a bias towards a higher representation of nucleotides A and T, which leads to a subsequent bias in the corresponding encoded amino acids (Shen et al., 2009). The A + T composition of the first and second codon positions in the 13 PCGs in T. serrata is 60.0% and 64.0%, respectively, but that of the third codon positions elevates to 68.9%, which is lower than values from the other seven sessile barnacles. 3.3. Base composition and skew The bias of the base composition in each gene can be described by skewness (Perna and Kocher, 1995) that measures the relative numbers of As to Ts (AT skew) and Gs to Cs (GC skew), and is calculated as (A% − T%)/(A% + T%) and (G% − C%)/(C% + G%), respectively. The

heavy strand in the T. serrata mitochondrial genome consists of 34.9% A, 22.2% C, 12.5% G, and 30.5% T bases. AT and GC skews of the whole genome are 0.068 and −0.280, respectively. The A + T contents are 64.1% and 70.8% for srRNA and lrRNA, respectively, and those of 13 PCGs range from 58.7% (cox3) to 70.1% (nd4L). All 13 PCGs consist of 25.4% A, 19.2% C, 16.5% G, and 38.9% T bases. Except for the atp8 gene with AT skew = 0 (i.e., equal number of A and T), the other 12 PCGs and both rRNAs have skews of T vs. A (AT skew between −0.006 and −0.388). On the other hand, only four PCGs have skews of G vs. C (GC skew ranging from 0.298 to 0.327), and the majority of PCGs have skews of C vs. G (GC skew between −0.608 and −0.168). However, both rRNAs have skews of G vs. C (GC skews are 0.291 and 0.370 for srRNA and lrRNA, respectively) (Table 4).

3.4. Ribosomal and transfer RNA genes The lengths of srRNA and lrRNA are 825 and 1306 bp, respectively, which are similar to values in the other barnacle mitochondrial genomes (Shen et al., 2014a; Tsang et al., 2014c; Wares, 2015). The two rRNAs, located between trnL1 and the control region, are separated by one tRNA gene (trnV) (Fig. 1; Table 2). Both lrRNA and srRNA are encoded on the light strand, which is similar to the some other reported crustacean mitochondrial genomes (Shen et al., 2010, 2011, 2012a). There are 22 tRNA genes in the T. serrata mitochondrial genome, each folding into a clover-leaf secondary structure ranging in different sizes from 58 (trnS1) to 70 (trnS2) nucleotides (Table 2). The anticodon usage, gene length and A + T composition are similar to those of other barnacle mitochondrial genomes.

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Table 2 Mitochondrial genomic profile of T. serrata (Crustacea: Sessilia). Gene

Strand

cox1 trnL2 cox2 trnD atp8 atp6 cox3 trnG nd3 trnR trnN trnA trnE trnS1 trnF nd5 trnH nd4 nd4L trnP trnT nd6 cob trnS2 trnC trnY nd1 trnL1 lrRNA trnV srRNA control region trnK trnQ trnI trnM nd2 trnW a

H H H H H H H H H H H H H H L L L L L L H H H H H H L L L L L L L H H H H

Position

Nucleotides

Start

Stop

1 1551 1623 2307 2371 2523 3188 3975 4041 4393 4455 4518 4584 4650 4724 4788 6492 6556 7879 8173 8235 8299 8784 9999 10,069 10,137 10,255 11,182 11,250 12,556 12,622 13,447 13,836 13,933 14,000 14,069 14,134 15,131

1548 1618 2306 2370 2529 3188 3974 4040 4392 4454 4517 4583 4648 4707 4787 6491 6555 7885 8169 8235 8298 8784 9923 10,068 10,130 10,201 11,178 11,249 12,555 12,621 13,446 13,835 13,899 14,000 14,067 14,133 15,132 15,195

1548 68 684 64 159 666 787 66 352 62 63 66 65 58 64 1704 64 1330 291 63 64 486 1140 70 62 65 924 68 1306 66 825 389 64 68 68 65 999 65

Codons

Anti-codon

Start

Stop

ATA

TAA

2 4 0 0 −7 −1 0 0 0 0 0 0 1 16 0 0 0 −7 3 −1 0 −1 75 0 6 53 3 0 0 0 0 0 33 −1 1 0 −2 5

TAA ATG

TAA

ATA ATG ATG

TAA TAA T-

ATT

T-

GTC

TCC TCG GTT TGC TTC GCT GAA ATG

TAG

ATG ATA

TTAA

GTG

TGG TGT ATA ATG

TAA TAA TGA GCA GTA

ATA

TAG TAG TAC

TTT TTG GAT CAT ATG

Intergenic sequencea

TAA TCA

Negative numbers indicate overlapping nucleotides between adjacent genes.

3.5. Gene arrangement Comparison of gene arrangement may be a useful approach for phylogenetic analysis, especially when we focus on ancestral relationships (Boore and Brown, 1998). Pancrustaceans, comprising crustaceans and hexapods (von Reumont et al., 2012; Rota-Stabelli et al., 2013), exhibit an ancestral mitochondrial gene arrangement (Boore et al., 1998; Shen et al., 2007). The eight sessile barnacles share three conserved

gene blocks (trnM- nd2- trnW- cox1- trnL2- cox2- trnD- atp8- atp6cox3- trnG- nd3- trnR- trnN- trnA- trnE- trnS1, trnT- nd6- cob- trnS2, and nd1- trnL1- lrRNA- trnV- srRNA) (genes encoded on the light strand are underlined, same below), of which the former two are derived from the pancrustacean ground pattern. Of the eight Sessilia species, three species from families Archaeobalanidae (S. amaryllis), Pyrgomatidae (N. grandis) and Balanidae (A. amphitrite) have identical gene arrangement (Fig. 2).

Table 3 Codon usage in 13 mitochondrial PCGs of T. serrata (Crustacea: Sessilia). Codon

Number

Percent

Codon

Number

Percent

Codon

Number

Percent

Codon

Number

Percent

UUU-F UUC-F UUA-L UUG-L CUU-L CUC-L CUA-L CUG-L AUU-I AUC-I AUA-M AUG-M GUU-V GUC-V GUA-V GUG-V

245 115 170 116 115 57 88 27 220 84 142 68 121 38 48 24

6.64 3.12 4.61 3.14 3.12 1.55 2.39 0.73 5.96 2.28 3.85 1.84 3.28 1.03 1.30 0.65

UCU-S UCC-S UCA-S UCG-S CCU-P CCC-P CCA-P CCG-P ACU-T ACC-T ACA-T ACG-T GCU-A GCC-A GCA-A GCG-A

102 65 65 16 65 25 45 9 83 34 65 12 90 59 27 10

2.76 1.76 1.76 0.43 1.76 0.68 1.22 0.24 2.25 0.92 1.76 0.33 2.44 1.60 0.73 0.27

UAU-Y UAC-Y UAA-⁎ UAG-⁎

83 66 8 2 32 45 47 14 61 58 69 29 42 33 68 29

2.25 1.79 0.22 0.05 0.87 1.22 1.27 0.38 1.65 1.57 1.87 0.79 1.14 0.89 1.84 0.79

UGU-C UGC-C UGA-W UGG-W CGU-R CGC-R CGA-R CGG-R AGU-S AGC-S AGA-S AGG-S GGU-G GGC-G GGA-G GGG-G

29 6 82 19 19 4 27 10 24 15 66 0 102 17 91 42

0.79 0.16 2.22 0.52 0.52 0.11 0.73 0.27 0.65 0.41 1.79 0.00 2.76 0.46 2.47 1.14

* means stop codon.

CAU-H CAC-H CAA-Q CAG-Q AAU-N AAC-N AAA-K AAG-K GAU-D GAC-D GAA-E GAG-E

X. Shen et al. / Marine Genomics 22 (2015) 63–69 Table 4 Nucleotide composition and skews of T. serrata mitochondrial protein-coding and ribosomal RNA genes. Gene

atp6 atp8 cob cox1 cox2 cox3 nd1 nd2 nd3 nd4 nd4L nd5 nd6 srRNA lrRNA All PCGs H-strand

Proportion of nucleotides A

C

G

T

0.294 0.340 0.269 0.263 0.304 0.243 0.205 0.289 0.295 0.208 0.241 0.228 0.298 0.319 0.336 0.254 0.349

0.257 0.258 0.249 0.238 0.225 0.257 0.116 0.224 0.241 0.108 0.103 0.113 0.245 0.127 0.092 0.192 0.222

0.111 0.063 0.146 0.170 0.133 0.156 0.222 0.111 0.116 0.214 0.196 0.208 0.082 0.232 0.200 0.165 0.125

0.338 0.340 0.335 0.329 0.338 0.344 0.458 0.375 0.347 0.471 0.460 0.451 0.374 0.322 0.372 0.389 0.305

A+T (%)

AT skew

GC skew

63.2 67.9 60.4 59.2 64.2 58.7 66.2 66.5 64.2 67.8 70.1 67.9 67.3 64.1 70.8 64.3 65.4

−0.069 0.000 −0.109 −0.111 −0.052 −0.173 −0.382 −0.130 −0.080 −0.388 −0.314 −0.329 −0.113 −0.006 −0.051 −0.209 0.068

−0.396 −0.608 −0.259 −0.168 −0.257 −0.243 0.314 −0.337 −0.349 0.327 0.310 0.298 −0.497 0.291 0.370 −0.077 −0.280

With reference to the pancrustacean ground pattern, seven conserved genes blocks are found in their mitochondrial genomes, including cox1- trnL2- cox2, trnD- atp8- atp6- cox3- trnG- nd3, trnR- trnN, trnFnd5- trnH- nd4- nd4L, nd6- cob- trnS2, nd1- trnL1- lrRNA- trnV- srRNA and trnM- nd2- trnW. Moreover, translocations of at least six tRNAs (trnA, trnE/trnS2, trnP/trnT, trnK, trnQ and trnC) are identified, and translocation and inversion occur in one tRNA (trnY) simultaneously. Gene rearrangements are not only restricted to tRNAs, but also include PCGs as well in the two Megabalanus (M. ajax and M. volcano) mitochondrial genomes, in which an inversion of a large gene block (including 3 PCGs and 3 tRNAs) is identified. In the conserved gene arrangement shared by the other six sessile barnacles studied, the order of six genes is: trnF- nd5- trnH- nd4- nd4L- trnP, while an inversion of this block from the light strand to heavy strand is observed in the two Megabalanus species (trnP- nd4L- nd4- trnH- nd5- trnF). Comparison of gene orders in all Sessilia and the pancrustacean ground plan suggests that the arrangement of this gene block in Megabalanus is a derived character (Fig. 2). Thus, large-scale gene rearrangements are found in

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the eight sessile barnacle mitochondrial genomes as compared to the pancrustacean ground pattern (Fig. 2). In other words, mitochondrial gene order is not conserved within the order Sessilia and represents a potential informative marker for inferring phylogenetic relationships within the order and its phylogenetic position in Maxillopoda. More mitochondrial genome data are needed to shed further light towards Sessilia phylogeny. 3.6. Phylogeny of Sessilia To investigate the phylogenetic position and the inner relationships of the order Sessilia, phylogenetic trees were constructed with nucleotide sequences of 13 PCGs from 15 complete mitochondrial genomes of Maxillopoda. Both ML and BI analyses lead to identical tree topologies (Fig. 3). In the tree, the three species from Copepoda (T. japonicus, T. californicus and C. hyperboreus) cluster together with high support (BPM = 100, BPP = 1.00). A. americanus (Branchiura) clusters with A. armillatus (Pentastomida), and this clade groups with all ten cirriped species from Thecostraca (BPM = 100, BPP = 1.00). Within Cirripedia, the two stalked barnacle species from the order Scalpelliformes (C. mitella and P. polymerus) cluster together. Within Sessilia, A. amphitrite (Balanidae) cluster with S. amaryllis (Archaeobalanidae), and the two group with N. grandis (Pyrgomatidae). Since the other two members of Balanidae (M. ajax and M. volcano) do not group with A. amphitrite, the family does not constitute a monophyletic assemblage (Fig. 3). In addition, while the gene arrangements of A. amphitrite, S. amaryllis and N. grandis are identical, the two Megabalanus have large-scale rearrangements as discussed above (Fig. 2). Therefore, both phylogenetic analysis using PCG sequences and gene order comparison suggest that Balanidae is not monophyletic, which is also supported by a phylogenetic analysis of the mitochondrial rRNA sequences (data not shown). This is consistent with results from recent phylogenetic analyses based on nuclear rRNA and/or individual mitochondrial genes (Simon-Blecher et al., 2007; Perez-Losada et al., 2008, 2014; Tsang et al., 2014a). However, it should be noted that in these studies and the present one, the support values of internal branches are not very high and the exemplars from Balanidae were limited. Therefore, the non-monophyly of the family Balanidae needs to be elucidated further. It is hoped that a comprehensive phylogeny of Sessilia can be revealed when more mitochondrial genomic data become

Fig. 2. Linearized representation of the gene order arrangement in eight mitochondrial genomes from the order Sessilia (Crustacea: Maxillopoda). Gene segments are not drawn to scale. Shaded boxes refer to conserved gene blocks; orange and green colors indicate gene blocks with or without inversion, respectively as compared to the pancrustacean ground pattern. Genes that are encoded on the heavy and light strands are placed on the top and bottom lines, respectively.

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Fig. 3. Phylogenetic tree constructed from both the ML and Bayesian analyses of 13 PCGs (nucleotide acid data) from 15 mitochondrial genomes The numbers at the nodes indicate the bootstrap values obtained from ML analysis (left) and Bayesian posterior probabilities (right), respectively.

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