Morphology and mitochondrial genome-based analysis of the systematics and evolution of Acanthochitona species (Polyplacophora: Acanthochitonidae)

Abstract

Chitons, known as marine living fossils, have retained their ancestral traits for approximately 300 million years. The genus Acanthochitona (Polyplacophora: Acanthochitonidae), characterized by the presence of 9 pairs of sutural tufts on a well-expanded girdle, is distributed across the intertidal zones of South Korea, Japan, China, and the Indo-Pacific. This study examined five Acanthochitona species from South Korea: A. achates, A. circellata, A. defilippii, A. rubrolineata, and A. feroxa sp. nov. Their mitochondrial genome sequences ranged from 14,986 to 15,006 bp in length and with a gene content typical for Polyplacophora. Genetic (including a transitive consistency score [TCS] genetic network), principal coordinate, phylogenetic network, and CO1-based barcoding gap analyses confirmed a new species, A. feroxa sp. nov., which exhibited morphologically distinct dorsal spicules and radulae. Maximum likelihood (ML) and Bayesian inference (BI) trees were constructed based on the CO1 sequences of 28 polyplacophoran species belonging to 9 families, which placed these five Acanthochitona species within a monophyletic family, Acanthochitonidae. The analyses also indicated the polyphyletic nature of Mopaliidae, recommending a reclassification. Divergence time estimation revealed that Acanthochitona deviated during the Late Cretaceous (ca. 83.94 mya), with continued speciation occurring in the Paleogene and Neogene periods. Additionally, we constructed a pictorial key based on the ML tree for morphologically identifying the five Acanthochitona species. This study contributes to the understanding of speciation and phylogenetic relationships within the Acanthochitonidae, offering valuable insights into the classification scheme and mitochondrial genome evolution of chitons in the western Pacific.

Keywords

Chiton / Acanthochitona / Mitochondrial genome / Morphology / Molecular phylogeny / Divergence time

References

[1] Akintola AA, Park B, Choi EH, Hwang UW. Complete mitochondrial genome of a malaria vector mosquito Anopheles sinensis from South Korea. Mitochondr DNA B Resour, 2022, 7: 881-883

[2] Alnashiri H, Thomas L, Thaikkottathil M, Philip S, Kutty R, Sivanpillai S, Abo Rasain AM. Phylogenetic affinities of Acanthopleurinae Dall, 1889 chitons (Mollusca: Polyplacophora: Chitonidae) from Jazan Coast in the Red Sea and Western Indo-Pacific. J Mar Sci Eng, 2023, 11: 158

[3] Alnashiri H, Thomas L, Philip S, Thaikkottathil M, Sureshkumar S, Kutty R. Complete mitochondrial genome and phylogenetic relationships of the Red Sea chiton Acanthopleura vaillantii Rochebrune, 1882 (Polyplacophora: Chitonida). Thalassas, 2024, 40: 51-58

[4] Bonfitto A, Dell’Angelo B, Evangelisti F, Sabelli B. The genus Acanthochitona (Mollusca: Polyplacophora) in the Mediterranean Sea: morphological and molecular data. Sci Mar, 2011, 75: 171-180

[5] Boore JL. Sankoff D, Nadeau JH. The duplication/random loss model for gene rearrangement exemplified by mitochondrial genomes of deuterostome animals. Comparative Genomics. Computational Biology, 2000, Dordrecht, Springer133147

[6] Boore JL, Brown WM. Complete DNA sequence of the mitochondrial genome of the black chiton, Katharina tunicata. Genetics, 1994, 138: 423-443

[7] Bouckaert R, Heled J, Kühnert D, Vaughan T, Wu CH, Xie D, Suchard MA, Rambaut A, Drummond AJ. BEAST 2: a software platform for Bayesian evolutionary analysis. PLoS Comput Biol, 2014, 10: e1003537

[8] Castresana J. Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol Biol Evol, 2000, 17: 540-552

[9] Chan PP, Lowe TM. tRNAscan-SE: searching for tRNA genes in genomic sequences. Methods Mol Biol, 2019, 1962: 1-14

[10] Choi EH, Hwang UW. Complete mitochondrial genome of a golden orb-web spider Trichonephila clavata (Chelicerata, Arachnida) from South Korea. Mitochondr DNA B Resour, 2023, 8: 723-725

[11] Choi EH, Choi NR, Hwang UW. The mitochondrial genome of an endangered freshwater snail Koreoleptoxis nodifila (Caenogastropoda: Semisulcospiridae) from South Korea. Mitochondr DNA B Resour, 2021, 6: 1120-1123

[12] Choi EH, Yeo MY, Kim G, Park B, Shin CR, Baek SY, Hwang UW. Liolophura species discrimination with geographical distribution patterns and their divergence and expansion history on the northwestern Pacific coast. Sci Rep, 2021, 11: 17602

[13] Clement M, Snell Q, Walke P, Posada D, Crandall K. TCS: estimating gene genealogies. Parallel and distributed processing symposium, international, 2002, USA, IEEE Computer Society1-7

[14] Dierckxsens N, Mardulyn P, Smits G. NovoPlasty: de novo assembly of organelle genomes from whole genome data. Nucleic Acids Res, 2017, 45: e18

[15] Donath A, Jühling F, Al-Arab M, Bernhart SH, Reinhardt F, Stadler PF, Middendorf M, Bernt M. Improved annotation of protein-coding genes boundaries in metazoan mitochondrial genomes. Nucleic Acids Res, 2019, 47: 10543-10552

[16] Dowton M, Campbell NJH. Intramitochondrial recombination–is it why some mitochondrial genes sleep around?. Trends Ecol Evol, 2001, 16: 269-271

[17] Eernisse DJ, Reynolds PD. Harrison FW, Kohn AJ. Polyplacophora. Microscopic anatomy of invertebrates, 1994, New York, Wiley-Liss561105

[18] Eernisse DJ, Draeger A, Pilgrim EM. Chitons (Mollusca: Polyplacophora) rafted on tsunami debris from Japan to the shores of Washington, Oregon, and Hawai'i. Aquat Invasions, 2018, 13: 71-86

[19] Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit i from diverse metazoan invertebrates. Mol Mar Biol Biotechnol, 1994, 3: 294-299

[20] Giribet G, Okusu A, Lindgren AR, Huff SW, Schrödl M, Nishiguchi MK. Evidence for a clade composed of molluscs with serially repeated structures: monoplacophorans are related to chitons. Proc Natl Acad Sci U S A, 2006, 103: 7723-7728

[21] Grant JR, Enns E, Marinier E, Mandal A, Herman EK, Chen C, Graham M, Van Domselaar G, Stothard P. Proksee: in-depth characterization and visualization of bacterial genomes. Nucleic Acids Res, 2023, 51: W484-W492

[22] Guerra D, Bouvet K, Breton S. Mitochondrial gene order evolution in Mollusca: inference of the ancestral state from the mtDNA of Chaetopleura apiculata (Polyplacophora, Chaetopleuridae). Mol Phylogenet Evol, 2018, 120: 233-239

[23] Hall TA. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl Acids Symp Ser, 1999, 41: 95-98

[24] Hebert PD, Cywinska A, Ball SL, DeWaard JR. Biological identifications through DNA barcodes. Proc R Soc Lond Ser B-Biol Sci, 2003, 270: 313-321

[25] Hong JS, Dell’Angelo B, Van Belle RA. The chiton fauna (Mollusca: Polyplacophora) of Korea. Part II: Suborder Acanthochitonina. Korean J Zool, 1990, 33: 373-395

[26] Hong S, Park B, Kim G, Choi EH, Hwang UW. Possible species discrimination of a blotched nerite Nerita albicilla with their distribution pattern and demographic history in the Indo-Pacific. Sci Rep, 2023, 13: 4545

[27] Huson DH, Bryant D. Application of phylogenetic networks in evolutionary studies. Mol Biol Evol, 2006, 23: 254-267

[28] Ibáñez-Carvajal CM (2017) Evolutionary biogeography of the Southeastern Pacific polyplacophorans.

[29] Irisarri I, Eernisse DJ, Zardoya R. Molecular phylogeny of Acanthochitonina (Mollusca: Polyplacophora: Chitonida): three new mitochondrial genomes, rearranged gene orders and systematics. J Nat Hist, 2014, 48: 2825-2853

[30] Irisarri I, Uribe JE, Eernisse DJ, Zardoya R. A mitogenomic phylogeny of chitons (Mollusca: Polyplacophora). BMC Evol Biol, 2020, 20: 1-15

[31] Jörger KM, Meyer R, Wehrtmann IS. Species composition and vertical distribution of chitons (Mollusca: Polyplacophora) in a rocky intertidal zone of the Pacific coast of Costa Rica. J Mar Biol Assoc U K, 2008, 88: 807-816

[32] Kalyaanamoorthy S, Minh BQ, Wong TKF, Von Haeseler A, Jermiin LS. ModelFinder: fast model selection for accurate phylogenetic estimates. Nat Methods, 2017, 14: 587-589

[33] Kim G, Hwang UW. The population genetic structure and phylogeographic dispersal of Nodularia breviconcha in the Korean Peninsula based on COI and 16S rRNA genes. PLoS ONE, 2023, 18: e0288518

[34] Kim DW, Yoo WG, Park HC, Yoo HS, Kang DW, Jin SD, Min HK, Paek WK, Lim J. DNA barcoding of fish, insects, and shellfish in Korea. Genomics Inform, 2012, 10: 206-211

[35] Kim G, Jeong KC, Choi EH, Ryu SH, Lim YJ, Jun J, Lee YS, Hwang UW. The complete mitochondrial genome of an Asian crested ibis Nipponia Nippon (Pelecaniformes, Threskiornithidae) from South Korea. Mitochondr DNA B Resour, 2019, 4: 3707-3708

[36] Kim IH, Shin CR, Kim G, Park B, Kim KB, Choi EH, Hwang UW. Complete mitochondrial genome of Acanthochitona defilippii (Polyplacophora: Chitonida) from South Korea. Mitochondr DNA B Resour, 2024, 9: 1029-1033

[37] Laghi GF. Upper Triassic chitons from the Italian dolomites. Lav Soc Venez Cienc Nat, 2005, 30: 79-84

[38] Laslett D, Canbäck B. ARWEN: a program to detect tRNA genes in metazoan mitochondrial nucleotide sequences. Bioinform, 2008, 24: 172-175

[39] Lee JH, Choi EH, Kim SK, Ryu SH, Hwang UW. Mitochondrial genome of the cockscomb pearl mussel Cristaria plicata (Bivalvia, Unionoida, Unionidae). Mitochondr DNA, 2012, 23: 39-41

[40] Letunic I, Bork P. Interactive tree of life (iTOL) v6: recent updates to the phylogenetic tree display and annotation tool. Nucleic Acids Res, 2024, 52: W78-W82

[41] Madeira F, Pearce M, Tivey ARN, Basutkar P, Edbali O, Madhusoodanan N, Kolesnikov A, Lopes R. Search and sequence analysis tools services from EMBL-EBI in 2022. Nucleic Acids Res, 2022, 50: W276-W279

[42] Mikkelsen NT, Kocot KM, Halanych KM. Mitogenomics reveals phylogenetic relationships of caudofoveate aplacophoran molluscs. Mol Phylogenet Evol, 2018, 127: 429-436

[43] Montoya J, Ojala D, Attardi G. Distinctive features of the 5′-terminal sequences of the human mitochondrial mRNAs. Nature, 1981, 290: 465-470

[44] Ni G, Kim T, Shin Y, Park J, Lee Y, Kil HJ, Park JK. Life-history features and oceanography drive phylogeographic patterns of the chiton Acanthochitona cf. rubrolineata (Lischke, 1873) in the northwestern Pacific.. PeerJ, 2020, 8: e8794

[45] Ojala D, Montoya J, Attardi G. tRNA punctuation model of RNA processing in human mitochondria. Nature, 1981, 290: 470-474

[46] Okusu A, Schwabe E, Eernisse DJ, Giribet G. Towards a phylogeny of chitons (Mollusca, Polyplacophora) based on combined analysis of five molecular loci. Org Divers Evol, 2003, 3: 281-302

[47] Osca D, Irisarri I, Todt C, Grande C, Zardoya R. The complete mitochondrial genome of Scutopus ventrolineatus (Mollusca: Chaetodermomorpha) supports the Aculifera hypothesis. BMC Evol Biol, 2014, 14: 1-10

[48] Owada M. Radula and shell microstructure variations are congruent with a molecular estimate of shallow-water Japanese chitons. Zool Sci, 2023, 40: 390-403

[49] Palumbi SR, Martin A, Romano S, McMillan WO, Stice L, Grabowski G. The simple fool’s guide to PCR. Dept of Zoology, 1991, Honolulu, University of Hawaii

[50] Park B, Choi EH, Kim G, Shin CR, Hwang J, Baek SY, Hwang UW. The complete mitochondrial genome of the two-spotted cricket Gryllus bimaculatus (Orthoptera: Gryllidae) from South Korea. Mitochondr DNA B Resour, 2021, 6: 1144-1146

[51] Park J, Lee Y, Kim T, Kern E, Kil HJ, Eernisse DJ, Saito H, Park JK. Molecular survey of Cryptoplax japonica (Polyplacophora: Cryptoplacidae) reveals cryptic lineages in the northwestern Pacific.. J Molluscan Stud, 2022, 88: eyac016

[52] Perrier X, Jacquemoud-Collet JP (2006) DARwin software: Dissimilarity analysis and representation for windows. Available at: http://darwin.cirad.fr/Darwin

[53] Pojeta JJ, Eernisse DJ, Hoare RD, Henderson MD. Echinochiton dufoei: a new spiny Ordovician chiton. J Paleontol, 2003, 77: 646-654

[54] Puchalski SS, Eernisse DJ, Johnson CC. The effect of sampling bias on the fossil record of chitons (Mollusca, Polyplacophora). Am Malacol Bull, 2008, 25: 87-95

[55] Puillandre N, Brouillet S, Achaz G. ASAP: assemble species by automatic partitioning. Mol Ecol Resour, 2021, 21: 609-620

[56] Rambaut A (2018) FigTree, version 1.4. 4, Accessed November 26

[57] Ríos CIG, Pérez NMP, López JF, Fuentes FA. Calibrating the chitons (Mollusca: Polyplacophora) molecular clock with the mitochondrial DNA cytochrome C oxidase I gene. Rev Biol Mar Oceanogr, 2014, 49: 193-207

[58] Ronquist F, Teslenko M, Van Der Mark P, Ayres DL, Darling A, Höhna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP. MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst Biol, 2012, 61: 539-542

[59] Rozas J, Ferrer-Mata A, Sánchez-DelBarrio JC, Guirao-Rico S, Librado P, Ramos-Onsins SE, Sánchez-Gracia A. DnaSP 6: DNA sequence polymorphism analysis of large data sets. Mol Biol Evol, 2017, 34: 3299-3302

[60] Rubinoff D. Utility of mitochondrial DNA barcodes in species conservation. Conserv Biol, 2006, 20: 1026-1033

[61] San Mauro D, Gower DJ, Zardoya R, Wilkinson M. A hotspot of gene order rearrangement by tandem duplication and random loss in the vertebrate mitochondrial genome. Mol Biol Evol, 2006, 23: 227-234

[62] Scherholz M, Redl E, Wollesen T, Todt C, Wanninger A. Aplacophoran mollusks evolved from ancestors with polyplacophoran-like features. Curr Biol, 2013, 23: 2130-2134

[63] Schwabe E. A catalogue of Recent and fossil chitons (Mollusca: Polyplacophora). Addenda. Novapex, 2005, 6: 89-105

[64] Shin CR, Choi EH, Kim G, Baek SY, Park B, Hwang J, Jun J, Kil HJ, Oh H, Lee K, Kim SH, Lee J, Suh SJ, Park D, Suk HY, Lee YS, Lee YS, Hwang UW. Characterization of metapopulation of Ellobium chinense through Pleistocene expansions and four covariate COI guanine-hotspots linked to G-quadruplex conformation. Sci Rep, 2021, 11: 12239

[65] Sigwart JD, Schwabe E. Anatomy of the many feeding types in polyplacophoran molluscs. Invert Zool, 2017, 14: 205-216

[66] Sigwart JD, Stoeger I, Knebelsberger T, Schwabe E. Chiton phylogeny (Mollusca: Polyplacophora) and the placement of the enigmatic species Choriplax grayi (H. Adams & Angas). Invertebr Syst, 2013, 27: 603-621

[67] Sirenko BI. New outlook on the system of chitons (Mollusca: Polyplacophora). Venus, 2006, 65: 27-49

[68] Sirenko BI. Four new species and one new genus of Jurassic chitons (Mollusca: Polyplacophora: Lepidopleurida) from the middle Russian Sea. Proc Zool Inst RAS, 2013, 317: 30-44

[69] Smith AB, Gale AS, Monks NE. Sea-level change and rock-record bias in the Cretaceous: a problem for extinction and biodiversity studies. Paleobiology, 2001, 27: 241-253

[70] Stebbins TD, Eernisse DJ. Chitons (Mollusca: Polyplacophora) known from benthic monitoring programs in the Southern California Bight. Festivus, 2009, 41: 53-100

[71] Strack HL, Leotta L. Additional data on Acanthochitona pilosa Schmidt-Petersen, Schwabe & Haszprunar, 2015 (Polyplacophora: Acanthochitonidae). Basteria, 2024, 88: 129-138

[72] Sun D, Lin Z, Teng X, Xu L, Qian L, Yu X, Wu H, Wang Z, Jin L, Liu X, Wang L, Qu J, Wang X, Xing Z. The complete mitochondrial genome and phylogenetic analysis of Lepidozona coreanica (Reeve, 1847). Mitochondr DNA B Resour, 2023, 8: 629-633

[73] Thompson JD, Higgins DG, Gibson TJ. CLUSTAL w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res, 1994, 22: 4673-4680

[74] Trifinopoulos J, Nguyen LT, von Haeseler A, Minh BQ. W-IQ-TREE: a fast online phylogenetic tool for maximum likelihood analysis. Nucleic Acids Res, 2016, 44: W232-W235

[75] Van Belle RA. The systematic classification of the chitons (Mollusca: Polyplacophora). Inf Soc Belge Malacol, 1983, 11: 1-164

[76] Veale AJ, Williams L, Tsai P, Thakur V, Lavery, . The complete mitochondrial genomes of two chiton species (Sypharochiton pelliserpentis and Sypharochiton sinclairi) obtained using Illumina next generation sequencing. Mitochondrial DNA A DNA Mapp Seq Anal, 2016, 27(1): 537-538

[77] Vendrasco MJ, Fernandez CZ, Eernisse DJ, Runnegar B. Aesthete canal morphology in the Mopaliidae (Polyplacophora). Am Malacol Bull, 2008, 25: 51-69

[78] Vinther J, Sperling EA, Briggs DE, Peterson KJ. A molecular palaeobiological hypothesis for the origin of aplacophoran molluscs and their derivation from chiton-like ancestors. Proc Biol Sci, 2012, 279: 1259-1268

[79] Vinther J, Parry L, Briggs DE, Van Roy P. Ancestral morphology of crown-group molluscs revealed by a new Ordovician stem aculiferan. Nature, 2017, 542: 471-474

[80] Vončina K, Mikkelsen NT, Morrow C, Ang R, Sigwart JD. Clarification of the taxonomic status of Acanthochitona discrepans (Brown, 1827) with new data for the North-East Atlantic Acanthochitona (Polyplacophora, Acanthochitonidae). Biodivers Data J, 2023, 11: e109554

[81] Watters GT. Some aspects of the functional morphology of the shell of infaunal bivalves (Mollusca), 1990, Columbus, The Ohio State University

[82] Wright D, Bishop JM, Matthee CA, von der Heyden S. Genetic isolation by distance reveals restricted dispersal across a range of life histories: implications for biodiversity conservation planning across highly variable marine environments. Divers Distrib, 2015, 21: 698-710

[83] Zhang J, Kapli P, Pavlidis P, Stamatakis A. A general species delimitation method with applications to phylogenetic placements. Bioinform, 2013, 29: 2869-2876

Funding

Kyungpook National University

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