The Mitochondrial Genomes of Pecten Albicans and Pecten Maximus (Bivalvia: Pectinidae) Reveal a Novel Gene Title Arrangement with Low Genetic Differentiation
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The mitochondrial genomes of Pecten albicans and Pecten maximus (Bivalvia: Pectinidae) reveal a novel gene Title arrangement with low genetic differentiation Author(s) Marín, Alan; Fujimoto, Takafumi; Arai, Katsutoshi Biochemical Systematics and Ecology, 61, 208-217 Citation https://doi.org/10.1016/j.bse.2015.06.015 Issue Date 2015-08 Doc URL http://hdl.handle.net/2115/66898 © 2015. This manuscript version is made available under the CC-BY-NC-ND 4.0 license Rights http://creativecommons.org/licenses/by-nc-nd/4.0/ Rights(URL) http://creativecommons.org/licenses/by-nc-nd/4.0/ Type article (author version) File Information all.pdf Instructions for use Hokkaido University Collection of Scholarly and Academic Papers : HUSCAP 1 The mitochondrial genomes of Pecten albicans and 2 Pecten maximus (Bivalvia: Pectinidae) reveal a novel 3 gene arrangement with low genetic differentiation 4 5 Alan Marín* Takafumi Fujimoto2 3 6 Hokkaido University,• Graduate School• Katsutoshi of Fisheries Arai Sciences, 3-1-1 Minato, 7 Hakodate, Hokkaido, 041-8611, Japan. 8 9 10 *Correspondente-mail: author 11 [email protected])6444 1955 12 Tel.:2 +81( 13 [email protected] 14 [email protected] 15 16 17 18 19 20 21 22 23 24 25 26 27 1 28 Abstract 29 30 Scallops (Bivalvia: Pectinidae) comprise more than 350 extant taxa including 31 species of high economic importance. However, its phylogenetic classification 32 remains unclear and so far only 7 scallop mitogenomes have been determined.Pecten In 33 albicansthis study, thePecten mitochondrial maximus genomes of two congeneric scallop species 34 - and atp8 were determined. Both mitogenomes- containRNA 12 35 protein coding genes ( is missing), two ribosomal genes,Pecten 20 22 transfer 36 genes, all encoded in the same strand. Overall, both mitogenomes are 37 highly similar with high nucleotide (93 %) and amino acid (98.7 %) sequence 38 identity. Both mitogenomes also contain the same gene order arrangement, 39 which is a novel contribution Pecten within Pectinidae. The highly similar mitochondrial 40 organization between both species suggested- a recent speciation information event. 41 Phylogenetic analysis based on complete protein coding gene as 42 well as large synteny Pectengene blocks confirmed the sister groupArgopecten relationship 43 between the genera (subfamily Pectininae) and (tribe Aequipectini). 44 45 Keywords 46 • Mitochondrial genome • Pectinidae • Synteny • Phylogenetics • 47 Amino acids • Scallops 48 49 50 2 51 1. Introduction 52 - 53 Metazoan mitochondrial genome-17 is typically built of double stranded 54 circular- DNA molecules of about 15 kb and usually encodes for 37 RNAgenes: 13 55 protein coding genes (PCGs), 2 ribosomal RNA genes and 22 transfer s that 56 are indispensable for protein translation (Boore, 1999; Liu et al., 2013). 57 Mitochondrial genes are considered important tools for evolution ary and 58 phylogenetic studies because of its high mutation rates, maternal transmission 59 (with exceptions in some animal groups) and the abundance of mitochondrial 60 copies in cells. In most metazoan taxa, gene order is usually well conserved. 61 However, marine bivalves are a notable exception to the common metazoan 62 mitochondrial constitution. Marine bivalve mitogenomes are known for their 63 high levels of gene rearrangement (Dreyer and Steiner, 2006; Yuan et al., 2012), 64 even among species of the same family (i.e. Pectinidae; Xu et al., 2010). An extra 65 large mitogenome size is another unusual characteristicPlacopecten of marine magellanicus bivalves. From 66 all Pectinidae species with availableank mitogenome, has 67 the largest size (32115 bp; GenB access number DQ088274).tRNAs The or presence tRNA-like of 68 tandem repeats elements and transposition involving - 69 structures are responsible for the large size and length variation (30 40 kb) 70 among individuals (La Roche et al., 1990; Smith and Snyder, 2007)atp8. Another 71 commontRNAs characteristic in most bivalve mitogenomes is the lack of gene and certain (Smith and Snyder, 2007). 72 73 The family Pectinidae comprises about 350 living species (Biscotti et al., 74 2007). In spite of the large fossil record, the phylogenetic relationships among scallop species are still unresolved. With 15 recognized species (World Register 3 75 ) 76 of MarinePecten Species, WORMS; http://www.marinespecies.org , scallops of the 77 genus represent- commercially important species distributed throughout 78 temperate and sub tropical seas in distant geographic areas such as Europe, 79 PectenAfrica, albicansAsia and AustraliaPecten ( Duncanmaximus and Wilson, 2012; Saavedra and Peña, 2004). 80 and constitute important fisheries (Saavedra and 81 Peña, 2004) as well. as interesting evolutionaryP. modelsalbicans due to their unknown 82 divergence times The Japanese baking scallop occurs fromP. maximus southern 83 Hokkaido to Kyushu in Japan (Okutani, 2000). On the other hand, is 84 distributed along the Northeast Atlantic from Norway down to North Africa 85 (Beaumont and Gjedrem, Pecten2007).To date, little is known about the mitochondrial 86 genome composition of species and few studies have addressed the 87 extremely low genetic differentiation among species of this genus, using only 88 partial mitochondrial gene fragments (Saavedra and Peña, 2004). It has been 89 shown that phylogenetic analysis based on complete mitogenome sequence data 90 have proved to produce much robust phylogenetic reconstructions enhancing 91 resolution and statistical confidence of inferred phylogenetic trees when 92 compared with analyses based only on smaller portions of the mtDNA (Ingman et al., 2000 and Yuan et al., 2012). 93 mito 94 Before this study, only seven complete genomes from thefrom family 2 95 Pectinidae were available in GenBankMizuhopecten database includingyessoensis 5 Chlamysspecies farreri 96 Mimachlamyssubfamilies named nobilis Chlamydinae Mimachlamys ( senatoria , P., 97 magellanicus and ) andArgopectenPalliolinae: irradians ( 98 Argopecten), and purpuratustwo species) from the tribe Aequipectini ( and . This is the first report on the complete 4 99 100 P.mitochondrial albicans codingP. maximus region and gene rearrangement of two Pectininae species: 101 and . The results of the present study will help to get 102 further insights into the very complex taxonomy and evolutionary relationships among Pectinidae species. 103 2. Materials and Methods 104 2.1. Sample collection and genomic DNA extraction 105 P. albicans P. maximus Oki 106 One individual of and were collected from 107 Island in Japan and Rye Bay in England, respectively. The- adductor muscles were 108 dissected and preserved in 95% ethanol -and stored at 20 °C. Genomic DNA waso a 109 extracted following 5 the standard phenol chloroform protocol and adjusted t concentration of 0 ng/μl and used for PCR. 110 2.2. Long PCR amplification and sequencing 111 112 Two long PCR primer sets were designed to amplify the entire 113 mitogenome in two fragments with overlapping segments, covering the complete 114 circular mitochondrial gen922ome. The9 primer insets P. albicans12SpectUF andP. maximusPecnaXX 115 (amplified fragments of 9 and 932 bp ofand , 116 respectivelyin P. albicans) and Pectco3F P. maximus and Pect12SR (amplified fragments approx. 13 and- 117 15 kb and , respectively) were cox3designednad2 based on multi 118 alignment comparisons of the mitochondrial 12S rRNA, and genes from 119 Pectinidae species retrieved from GenBank database (primer sequencesa LA Taqare 120 listed in Table 1). Large PCR products were obtained using TaKaR 121 polymerase (TaKaRa, Japan). To quicken-6 the sequencing process, additional shorter overlapping PCR fragments (2 kb) were amplified (see Table 1 for 5 122 - Full 123 primer list) using the large PCR fragments (10 15kb) as DNA templates. 124 length of both scallop mitogenomes were estimated based in the nucleotide size 125 content of the first large PCR product) amplified by primers 12SpectUF and 126 PecnaXX (which was fully sequenced plus the combination of overlapping large 127 and small PCR fragments from the "second half" of the mitogenome, which were 128 estimated based on comparison with molecular ladders of different sizes by 129 agarose electrophoresis. All PCR amplifications were performed on an ASTEC2 130 PC816 thermal cycler in a total volume of 50 μl, containing2 26.5 μl sterile H O, 5 2+ 131 μl 10 × LA PCR Taq buffer (Mg free, TaKaRa), 5 μlTaq MgCl (25mM), 8 μld NTPs 132 (2.5mM each), 2 μl each primer (10 μM), 0.5 μl LA polymerase (5 units/μl, 133 TaKaRa) and 1 μl of template DNA. Thermal cycling conditions were as follows: 134 initial denaturation for 2 min at 98 °C, followed by 35 cycles of denaturation for 135 20 s at 94 °C, annealing for 35 s at 60 °C, and extension for 10 min at 68 °C, 136 followed by a final extension for 10 min at 72 °C. Amplicons- were 137 electrophoresed in 1% agarose gels, stained with ethidium bromide, and 138 visualized under UV light. Subsequently, PCR products were extracted and 139 purified using the QIAquick Gel Extraction kit (Quiagen, Hilden, Germany), 140 directly sequenced in both directions by primer walking method on an ABI PRISM 3130XL Genetic Analyzer (Applied Biosystems, Hitachi, Japan). 141 - P. albicans P. maximus 142 The largest non coding region of the and 143 mitogenomes is characterized for the presence of long tandems repeats (1.6 kb 144 approx; Rigaa et al., 1995) and homopolymer runs, which affected the PCR 145 reaction and DNA sequencing process by polymerase slippage. Regrettably, a fragment containing the control region was not determined (approx. 6 and 7.5 kb 6 146 P. albicans P. maximus 147 in size in and , respectively). Thus, only the complete mitochondrial coding region is reported for both species. 148 nad1-rrnL-cox1 149 Additionally, to identify the gene cluster in more scallopnad1 150 species,cox1a highly conserved fragment includingrrnL, trn Rpartial trnM sequence of genes 151 and flanking the complete genes and Amusiumwere amplified pleuronectes and 152 Ylistrumsequenced japonicum in six additionalAnnachlamys scallop speciesmacassarensisincludingBractechlamys vexillum, 153 Decatopecten radula , Mimachlamys sanguinea , , 154 Mar and .