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Molecular Phylogenetic Relationships of Halacarid Mites Suggest the Reevaluation of Traditional Subfamily Classification Hiroko Meguro, Masahiro A

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Molecular Phylogenetic Relationships of Halacarid Mites Suggest the Reevaluation of Traditional Subfamily Classification Hiroko Meguro, Masahiro A Molecular phylogenetic relationships of halacarid mites suggest the reevaluation of traditional subfamily classification Hiroko Meguro, Masahiro A. Iwasa, Koichi Goka, Hiroshi Abé To cite this version: Hiroko Meguro, Masahiro A. Iwasa, Koichi Goka, Hiroshi Abé. Molecular phylogenetic relationships of halacarid mites suggest the reevaluation of traditional subfamily classification. Acarologia, Acarologia, 2017, 57 (3), pp.633-641. 10.24349/acarologia/20174182. hal-01535807 HAL Id: hal-01535807 https://hal.archives-ouvertes.fr/hal-01535807 Submitted on 9 Jun 2017 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Distributed under a Creative Commons Attribution - NonCommercial - NoDerivatives| 4.0 International License ACAROLOGIA A quarterly journal of acarology, since 1959 Publishing on all aspects of the Acari All information: http://www1.montpellier.inra.fr/CBGP/acarologia/ [email protected] Acarologia is proudly non-profit, with no page charges and free open access Please help us maintain this system by encouraging your institutes to subscribe to the print version of the journal and by sending us your high quality research on the Acari. Subscriptions: Year 2017 (Volume 57): 380 € http://www1.montpellier.inra.fr/CBGP/acarologia/subscribe.php Previous volumes (2010-2015): 250 € / year (4 issues) Acarologia, CBGP, CS 30016, 34988 MONTFERRIER-sur-LEZ Cedex, France The digitalization of Acarologia papers prior to 2000 was supported by Agropolis Fondation under the reference ID 1500-024 through the « Investissements d’avenir » programme (Labex Agro: ANR-10-LABX-0001-01) Acarologia is under free license and distributed under the terms of the Creative Commons-BY-NC-ND which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited. Acarologia 57(3): 633–641 (2017) DOI: 10.24349/acarologia/20174182 Molecular phylogenetic relationships of halacarid mites suggest the reevaluation of traditional subfamily classification Hiroko MEGURO1, Masahiro A. IWASA2 B, Koichi GOKA3 and Hiroshi ABÉ1 (Received 28 August 2016; accepted 01 November 2016; published online 16 May 2017; edited by Maria NAVAJAS) 1 Biological Laboratory, Graduate School of Bioresource Sciences, Nihon University, Kameino 1866, Fujisawa, Kanagawa Pref., Japan. [email protected], [email protected] 2 Laboratory of Wildlife Science, College of Bioresource Sciences, Nihon University, Kameino 1866, Fujisawa, Kanagawa Pref., Japan. [email protected] (B); Tel & Fax: +81-466-84-3745 3 National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-0053, Japan. [email protected] ABSTRACT — Molecular phylogenetic relationships were examined for marine mites in the subfamilies Halacarinae, Copidognathinae, Rhombognathinae and Halixodinae from the aquatic mite family Halacaridae by using nuclear 18S and 28S ribosomal RNA gene sequences. The analysis revealed incongruences between the clustering obtained in the phylogenetic trees and the current morphological classification in the subfamilies Halacarinae and Rhombognathinae (based on 18S sequences) and in the Rhombognathinae (based on both 28S and combined 18S and 28S sequences). By contrast, the tree clustering was consistent with the morphological taxonomic positions in the Copidognathinae and Halixodinae subfamilies. It can be concluded that molecular phylogenetic analysis challenges the current classifications of Halacarinae and Rhombognathinae subfamilies. Accordingly diagnostic characters for their classification should be reevaluated. KEYWORDS — Halacaridae; subfamily; DNA sequences; taxonomy; phylogeny INTRODUCTION ganisms. Recent molecular studies on mite tax- onomy have raised conflicts between morphology- The classification of mites has relayed mainly and molecular-based relationships challenging mite on morphological criteria, particularly exoskeletal classifications (Cruickshank 2002; Pepato et al. 2010; characteristics (Krantz 1978; Walter and Proctor Li et al. 2014; Matsuda et al. 2014), but have also 1999). The classification was constructed tradition- highlighted their value in evaluating mite classifica- ally, based only on the morphology-based relation- tions on the basis of reliable phylogenetic relation- ships of mites. However, it is unclear whether such ships. morphological characters reflect real evolutionary processes, partly because the traditional method in- The vast number of Prostigmata mites, halacarid evitably includes arbitrary choices of the morpho- mites (Halacaridae) are aquatic taxa which mostly logical characters used. In addition, it is some- inhabit coastal sea areas (Abé 1990). Since the 1800s times difficult to find structural homology to be the classification of halacarid mites was performed used for morphological comparisons between or- by using morphological criteria (e.g., Bartsch 2015). http://www1.montpellier.inra.fr/CBGP/acarologia/ 633 ISSN 0044-586-X (print). ISSN 2107-7207 (electronic) Meguro H. et al. So far approximately 1,300 species in 64 genera the body using needles and gently squeezed out in- have been described worldwide and there still re- ner contents from the body exoskeleton in the ly- main a large number of undescribed species. On sis – pK solution on the cap. The cap loaded with the other hand, studies on the phylogenetic rela- the mite body exoskeleton (idiosoma with legs), the tionships among halacarid taxa are scarce. Phylo- gnathosoma, and the inner body contents was care- genetic studies based on morphological characters fully placed back in the microtube with the solution, have been carried out only on Rhombognathinae and the microtube, with the cap attached, was spun mites which consist on of four genera: Rhombog- down at 12,000 rpm for a few seconds. Fresh 25 nathus, Rhombognathides, Isobactrus and Metarhom- µl nuclei lysis solution and 5 µl of 1% pK solution bognathus (Abé 1998, 2001a, b; Abé and Bartsch were added into the microtube that contained the 2007; Bartsch 2003, 2008, 2010). Only few studies idiosoma with legs, the gnathosoma, and the inner reported on their classification (Abé 2001b; Bartsch contents with the primary solution, and all of the 2015). Thereafter, halacarid mites have been used contents were incubated again at 50 °C for 120 min as partial information in the molecular phylogenetic and then at 95 °C for 20 min. After incubation, the analyses of acariform mites (Otto and Wilson 2001; exoskeleton and the gnathosoma were picked up for Pepato et al. 2010; Pepato and Klimov 2015; Dabert specimen preparation. Finally, after incubation, the et al. 2016). extract was diluted to 10% of its original concentra- tion using TE buffer (0.001 M EDTA, 0.01 M Tris- In this study, we analyzed nuclear riboso- HCl [pH 8.0]) following the method of Goka et al. mal gene (18S and 28S) sequences of several ha- (2001, 2009) and used as the source of the DNA tem- lacarid taxa: Copidognathinae (Copidognathus), Ha- plate for PCR amplification. lacarinae (Halacarus, Agauopsis and Thalassarachna), Limnohalacarinae (Limnohalacarus), Halixodinae (Bradyagaue and Agaue) and Rhombognathinae Fragments of the nuclear 18S and 28S ribosomal (Rhombognathus, Metarhonbognathus and Rhombog- RNA gene (rDNA) regions were amplified by the nathides) and re-evaluated the typological classifica- PCR method using the following primer sets: HAL tion of the halacarid taxa by morphological criteria 18S_2F (5’-GTG TCT GCC TTA TCA ACT TTC GAT based on the current molecular findings. GG-3’) for the forward direction and HAL 18S_2R (5’-GCC CCC GTC TGT CCC TCT TAA TC-3’) for the reverse direction for 18S rDNA, and 28S_V (28ee MATERIALS AND METHODS in Hillis and Dixon 1991) for the forward direction and 28S_VI (reverse of 28v in Hillis and Dixon 1991) We collected individual halacarid mites (Table 1). for the reverse direction for 28S rDNA. PCR amplifi- After sufficient starvation (over six months), each cations were conducted in accordance with the pro- mite was stored in 70% EtOH at room temperature. cedure of Goka et al. (2001), with 2 µL of each tem- To remove the EtOH of specimen storage, each mite plate DNA in a total reaction volume of 50 µL. The was put into a microtube and air-dried at room tem- PCR reaction mix contained 0.2 mM of each dNTP, 2 perature for a few hours. After removal of the EtOH, mM MgCl2, 1.25 units of Taq DNA polymerase (Am- 15 µl of nuclei lysis solution (Wizard Genomic DNA pliTaq Gold), and 0.5 mM of each primer. All PCR Purification Kit, Promega) and 2 µl of 1% proteinase reagents were purchased from Perkin Elmer Ap- K solution (pK, Merck) were added into the micro- plied Biosystems. Conditions for the amplification tube (Goka et al. 2001), and it was incubated at were an initial denaturation for 9 min at 95 °C; 40 cy- 50 °C for 120 min to soften the mite body. Previ- cles of 30 s at 94 °C, 30 s at 44-54 °C, and 2 min at 72 ously, the hinge of a 1.5 ml microtube was cut to °C; and a final extension for 7 min at 72 °C. After the remove the cap. After heat treatment, the
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