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Development of a new set of PCR primers for eDNA metabarcoding decapod crustaceans
expand article infoTomoyuki Komai, Ryo O. Gotoh§, Tetsuya Sado§, Masaki Miya§
‡ Department of Zoology, Natural History Museum and Institute, Chiba, Japan
§ Department of Ecology and Environmental Sciences, Natural History Museum and Institute, Chiba, Japan
Open Access

Abstract

The Decapoda is one of the largest orders within the class Malacostraca, comprising approximately 14,000 extant species and including many commercially important species. For biodiversity monitoring in a non-invasive manner, a new set of PCR primers was developed for metabarcoding environmental DNA (eDNA) from decapod crustaceans. The new primers (herein named “MiDeca”) were designed for two conservative regions of the mitochondrial 16S rRNA gene, which amplify a short, hyper-variable region (153–184 bp, 164 bp on average) with sufficient interspecific variations. With the use of MiDeca primers and tissue-derived DNA extracts, we successfully determined those sequences (154–189 bp) from 250 species, placed in 186 genera and 65 families across the suborder Dendrobranchiata and 10 of the 11 infraorders of the suborder Pleocyemata. We also preliminarily attempted eDNA metabarcoding from natural seawater collected at Banda, Tateyama, the Pacific coast of central Japan and detected 42 decapod species including 34 and 8 species with sequence identities of > 98% and 80–98%, respectively. The results suggest the usefulness of eDNA metabarcoding with MiDeca primers for biodiversity monitoring of the decapod species. It appears, however, that further optimisation of primer sequences would still be necessary to avoid possible PCR dropouts from eDNA extracts.

Key Words

mitochondrial 16S rRNA gene, biodiversity monitoring, MiDeca, natural sea water

Introduction

Classical methods of biodiversity monitoring have been primarily based on the collection of specimens and subsequent morphology-based identification. Such biodiversity monitoring is costly and time-consuming and requires considerable expertise for various taxonomic groups. Recent technological developments in molecular ecology have provided a novel tool for species detection using DNA present in aquatic or terrestrial environments (environmental DNA or eDNA; Taberlet et al. 2012).

There are two major approaches to applying eDNA analysis: “eDNA barcoding”, which aims at detecting a single species in the environment (species-specific approach); and “eDNA metabarcoding”, which simultaneously detects multiple species from an environmental sample (multi-species approach). The latter approach has been developed with rapidly developed high-throughput next-generation sequencing (NGS) (e.g. Taberlet et al. 2012, Thomsen et al. 2012, Miya et al. 2015, Valentini et al. 2016). Application of eDNA is now quite wide-ranging in studies of biodiversity, aquatic ecology and conservation biology (Bohmann et al. 2014, Díaz-Ferguson and Moyer 2014).

In particular, with regard to aquatic environments, the multi-specific assessment and monitoring of the fauna using eDNA have focused mainly on vertebrates (e.g. Thomsen et al. 2012, Kelly et al. 2014, Miya et al. 2015, Andruszkiewicz et al. 2017, Ushio et al. 2017, 2018), which are known to release abundant DNA derived from faeces, body mucus, blood and sloughed tissue or scales (Bohmann et al. 2014). The applicability of the eDNA metabarcoding for aquatic invertebrates, notably those with an exoskeleton (Crustacea), has received less attention (Thomsen et al. 2012, Rees et al. 2014). The vast majority of previous studies on crustaceans yielded a “species-specific” approach detecting invasive species (e.g. Tréguier et al. 2014, Dougherty et al. 2016, Larson et al. 2017) or monitoring seasonal migrations of particular species (Wu et al. 2018).

The Decapoda is the largest order of the crustacean class Malacostraca (Arthropoda: Pancrustacea), comprising more than 14,000 extant species worldwide (De Grave et al. 2009, Ahyong et al. 2011), with continuing discovery of new species. The great majority of decapod crustaceans are marine, but other environments have also been colonised, such as lowland freshwaters, mountain rivers, estuaries and even land. Decapods are also highly diverse in ecology and include a number of commercially important species (such as shrimp, prawn, lobster, crayfish, king crab, snow crab etc.), attracting much scientific and economic interests.

The importance of marker selection in eDNA metabarcoding has recently been emphasised (Coissac et al. 2012, Deagle et al. 2014). As there is no ideal universal metabarcode (Riaz et al. 2011), marker selection could be specific to the target taxonomic group. The aim of this study was to develop new universal PCR primers for eDNA metabarcoding of decapod crustaceans, which enable detection of multiple species for biodiversity assessment and monitoring. The performance of newly developed primers (herein named “MiDeca”) was tested using tissue-derived DNA extracts from 250 species and an eDNA sample from natural seawater collected from the near-shore environment.

Materials and methods

Primer development

Mitochondrial rRNA genes have been recommended for identification of animal taxa because they have a similar taxonomic resolution to the COI marker and they present conserved regions that flank variable regions, which allows the design of primers with high-resolving power for the target taxonomic group (Deagle et al. 2014). In order to identify a suitable region in the mitogenome for species identification based on eDNA, 267 whole mitogenome sequences of Decapoda registered in the databases have been downloaded from NCBI as of 17 October 2017.

Three requirements were considered for designing the new primers: 1) a target amplicon consisting of fewer than 200 bp is desirable because the eDNA will often be degraded; 2) the amplified regions include sufficient interspecific differences for all target species; and 3) conserved regions (20–30 bp) across all target species are located at both ends of the short hyper-variable region to simultaneously amplify the target sequences (Riaz et al. 2011, Miya et al. 2015, Valentini et al. 2016).

After removing problematic sequences (i.e. 18 sequences that could not be aligned with other sequences), the remaining 249 sequences from 203 species and five sequences of unidentified species of Cherax (Astacidea: Parastacidae) (Table 1) were subjected to multiple alignment using MUSCLE (Edgar 2004) implemented in MEGA7 (Kumar et al. 2016) with a default set of parameters. The aligned sequences were imported into MEGA7 for visual inspection of the conserved and hyper-variable regions. The visual search for a short hyper-variable region (up to 200 bp for paired-end sequencing using the Illumina MiSeq) flanked by two conservative regions (ca. 20–30 bp) was performed on the entire set of the two aligned 12S rRNA and 16S rRNA genes. Primers were designed using Primer3 (Rozen et al. 2000) accounting for G/C contents (40–60%) and melting temperature (Tm: 50–60 °C).

Mitogenome sequences used to design the MiDeca primers. Scientific names follow those as registered in GenBank database.

Suborder Infraorder Family Species Acc. No.
Dendrobranchiata Penaeidae Farfantepenaeus californiensis (Holmes, 1900) EU497054
Fenneropenaeus chinensis (Osbeck, 1765) DQ518969
Fenneropenaeus chinensis DQ656600
Fenneropenaeus indicus (H. Milne Edwards, 1837) KX462904
Fenneropenaeus merguiensis (de Man, 1888) KP637168
Fenneropenaeus penicillatus (Alcock, 1905) KP637169
Litopenaeus stylirostris (Stimpson, 1874) EU517503
Litopenaeus vannamei (Boone, 1931) DQ534543
Litopenaeus vannamei EF584003
Litopenaeus vannamei KT596762
Metapenaeopsis dalei (Rathbun, 1902) KU050082
Metapenaeus ensis (De Haan, 1844) KP637170
Parapenaeopsis hardwickii (Miers, 1878) KU899136
Penaeus monodon Fabricius, 1798 AF217843
Solenoceridae Solenocera crassicornis (H. Milne Edwards, 1837) KU899137
Sergestidae Acetes chinensis Hansen, 1919 JN689221
Pleocyemata Stenopodidea Stenopodidae Stenopus hispidus (Olivier, 1811) JN399096
Stenopus hispidus KC107811
Caridea Alpheidae Alpheus distinguendus de Man,1909 GQ892049
Alpheus lobidens De Haan, 1849 KP276147
Atyidae Caridina cf. nilotica HMG-2016 KU726823
Caridina gracilipes de Man, 1902 KM023648
Halocaridina rubra Holthuis, 1963 DQ917432
Halocaridina rubra KF437502
Halocaridina rubra KF437503
Halocaridina rubra KF437504
Halocaridina rubra KF437505
Halocaridina rubra KF437506
Halocaridina rubra KF437507
Halocaridina rubra KF437508
Halocaridina rubra KF437509
Halocaridinides fowleri Gordon, 1968 KX844723
Neocaridina denticulata (De Haan, 1844) JX156333
Paratya australiensis Kemp. 1917 KM978917
Stygiocaris lancifera Holthuis, 1960 KX844714
Stygiocaris stylifera Holthuis, 1960 KX844722
Typhlatya arfeae Jaume & Bréhier, 2005 KX844721
Typhlatya consobrina Botoşăneanu & Holthuis, 1970 KX844717
Typhlatya dzilamensis Alvarez, Iliffe & Villalobos, 2005 KX844719
Typhlatya galapagensis Monod & Cals, 1970 KX844711
Typhlatya galapagensis KX844718
Typhlatya garciai Chace, 1942 KX844720
Typhlatya iliffei Hart & Manning, 1981 KX844710
Typhlatya mitchelli Hobbs & Hobbs, 1976 KX844712
Typhlatya monae Chace, 1954 KX844715
Typhlatya pearsei Creaser, 1936 KX844709
Typhlatya sp. JR2016 KX844713
Typhlatya taina Estrada & Gómez, 1987 KX844708
Typhlopatsa pauliani Holthuis, 1956 KX844716
Alvinocarididae Alvinocaris chelys Komai & Chan, 2010 JX184903
Alvinocaris longirostris Kikuchi & Hashimoto, 1995 AB821296
Alvinocaris longirostris JQ035659
Nautilocaris saintlaurentae Komai & Segonzac, 2004 KF226726
Opaepele loihi Williams & Dobbs, 1995 JQ035657
Rimicaris exoculata Williams & Rona, 1986 KP284529
Rimicaris kairei Watabe & Hashimoto, 2000 JQ035656
Crangonidae Crangon hakodatei Rathbun, 1902 KU641481
Rhynchocinetidae Rhynchocinetes durbanensis Gordon, 1936 KT590405
Palaemonidae Macrobrachium bullatum Fincham, 1987 KM978918
Macrobrachium lanchesteri (de Man, 1911) FJ797435
Macrobrachium nipponense (De Haan, 1849) HQ830201
Macrobrachium rosenbergii (de Man, 1879) AY659990
Pleocyemata Caridea Palaemonidae Palaemon carinicauda (Holthuis, 1950) EF560650
Palaemon gravieri (Yu, 1930) KT935323
Palaemon gravieri KU899135
Palaemon serenus Heller, 1862 KM978916
Pandalidae Chlorotocus crassicornis (Costa, 1871) KY944589
Astacidea Astacidae Astacus astacus (Linnaeus, 1758) KX279347
Astacus astacus KX279348
Astacus leptodactylus Eschscholtz, 1823 KX279349
Astacus leptodactylus KX279350
Austropotamobius pallipes (Lerebullet, 1858) KP205430
Austropotamobius torrentium (Schrank, 1803) KX268734
Pacifastacus leniusculus (Dana, 1852) KX268740
Cambaridae Cambarus robustus Girard, 1852 KX268738
Orconectes sanbornii (Faxon, 1884) KU239995
Orconectes luteus (Creaser, 1933) KX268739
Orconectes rusticus (Girard, 1852) KU239994
Faxonius limosus (Rafinesque, 1817) KP205431
Procambarus acutus (Girard, 1852) KX268741
Procambarus alleni (Faxon, 1884) KT074363
Procambarus clarkii (Girard, 1852) JN991197
Procambarus clarkii JX316743
Procambarus clarkii KT036444
Procambarus clarkii KX268742
Procambarus fallax Hagen, 1870 KC107813
Procambarus fallax KT074364
Procambarus fallax KT074365
Cambaroididae Cambaroides dauricus Pallas, 1773 KX268735
Cambaroides japonicus De Haan, 1841 KX268736
Cambaroides schrenckii (Kessler, 1874) KX268737
Cambaroides similis (Koelbel, 1892) JN991196
Enoplometopidae Enoplometopus debelius Holthuis, 1983 KM488333
Enoplometopus occidentalis (Randall, 1840) KC107819
Nephropidae Homarus americanus H. Milne Edwards, 1837 HQ402925
Homarus gammarus (Linnaeus, 1758) KC107810
Metanephrops thomsoni (Bate, 1888) KP889215
Nephrops norvegicus (Linnaeus, 1758) LN681403
Parastacidae Astacopsis gouldi Clark, 1936 KM458973
Cherax bicarinatus (Gray, 1845) KM501041
Cherax boesemani Lukhaup & Pekny, 2008 KM501042
Cherax cainii Austin & Ryan, 2002 HG942366
Cherax cainii KF649849
Cherax cairnsensis Riek, 1969 HG799094
Cherax crassimanus Riek, 1967 HG942365
Cherax destructor Clark, 1936 AY383557
Cherax destructor HG799087
Cherax destructor HG799092
Cherax destructor HG799099
Cherax destructor HG942177
Cherax destructor HG942178
Cherax destructor KJ573468
Cherax destructor KJ573469
Cherax dispar Riek, 1951 HG799093
Cherax glaber Riek, 1967 KF649852
Cherax holthuisi Lukhaup & Pekny, 2006 KM501039
Cherax monticola Holthuis, 1950 KF649851
Cherax preissii (Erichson, 1846) HG799097
Cherax quadricarinatus (von Martens, 1868) HG942364
Cherax quadricarinatus KF649850
Cherax quinquecarinatus HG799091
Cherax robustus Riek, 1961 HG799090
Cherax sp. HG-2014a HG799095
Cherax sp. HG-2014a HG799096
Cherax sp. HG-2014b HG799098
Cherax sp. HMG-2016 KX119167
Pleocyemata Astacidea Palaemonidae Cherax sp. YPL-2015 KM501043
Cherax tenuimanus Smith, 1912 KP205429
Engaeus cunicularius (Erichson, 1846) HG942173
Engaeus lengana Hortitz, 1990 KF546209
Engaeus lyelli Clark, 1936 HG799086
Engaeus quadrimanus Clark, 1936 LK391949
Engaeus sericatus Clark, 1936 LK391948
Engaewa subcoerulea Riek, 1967 KT946764
Engaewa walpolea Horwitz & Adams, 2000 KT946765
Euastacus armatus (von Martens, 1866) KP294310
Euastacus spinifer (Heller, 1865) KM458972
Euastacus yarraensis (McCoy, 1888) HG942176
Geocharax gracilis Clark, 1936 HG942174
Geocharax gracilis HG942175
Gramastacus insolitus Riek, 1972 KX148478
Gramastacus lacus McCormack, 2014 LK022684
Paranephrops planifrons White, 1842 LN681402
Tenuibranchiurus glypticus Riek, 1951 KM453741
Glypheidea Glypheidae Laurentaeglyphea neocaledonica (Richer de Forges, 2006) KU500619
Neoglyphea inopinata Forest & de Saint Laurent, 1975 KT984196
Neoglyphea inopinata KT984197
Axiidea Axiidae Calocaris macandreae Bell, 1853 KC107812
Callianassidae Callianassa ceramica Fulton & Grant, 1906 KU350630
Callianassa ceramica KU362925
Corallianassa coutierei (Nobili, 1904) KC107817
Nihonotrypaea harmandi (Bouvier, 1903) LC221567
Nihonotrypaea thermophila Lin, Komai & Chan, 2007 JN897380
Paraglypturus tonganus Ahn, Kim, Ju & Min, 2017 KJ820739
Trypaea australiensis (Dana, 1852) KM501040
Strahlaxiidae Neaxius acanthus (A. Milne Edwards, 1879) KC107821
Neaxius glyptocercus (von Martens, 1868) JN897379
Gebiidea Thalassinidae Thalassina kelanang Moh & Chong, 2009 JN897378
Upogebiidae Austinogebia edulis (Ngoc-Ho & Chan, 1992) JN897376
Austinogebia wuhsienweni (Yu, 1931) LC006054
Upogebia major (De Haan, 1841) JF793665
Upogebia major JN897377
Upogebia pusilla (Petagna, 1792) KC107815
Upogebia yokoyai Makarov, 1938 KM886610
Achelata Palinuridae Palinurellus wieneckii (de Man, 1881) KC847078
Panulirus homarus (Linnaeus, 1758) JN542716
Panulirus japonicus (von Siebold, 1824) AB071201
Panulirus ornatus (Fabricius, 1798) GQ223286
Panulirus ornatus HM446347
Panulirus stimpsoni Holthuis, 1963 GQ292768
Panulirus versicolor (Latreille, 1804) JQ320274
Panulirus versicolor KC107808
Sagmariasus verreauxi (H. Milne Edwards, 1851) AB859775
Scyllaridae Ibacus ciliatus (von Siebold, 1824) KM488334
Scyllarides latus (Latreille, 1803) KC107814
Thenus orientalis (Lund, 1793) LK391947
Polychelida Polychelidae Polycheles typhlops Heller, 1862 KC107818
Anomura Kiwaidae Kiwa tyleri Thatje et al., 2015 KY423514
Munididae Munida gregaria (Fbricius, 1793) KU521508
Munidopsidae Shinkaia crosnieri Baba & Williams, 1998 EU420129
Porcellanidae Neopetrolisthes maculatus (H. Milne Edwards, 1837) KC107816
Petrolisthes haswelli Miers, 1884 LN624374
Diogenidae Clibanarius infraspinatus (Hilgendorf, 1869) LN626968
Lithodidae Paralithodes brevipes (H. Milne Edwards & Lucas, 1848) AB735677
Paralithodes camtschaticus (Tilesius, 1815) JX944381
Paguridae Pagurus longicarpus Say, 1817 AF150756
Brachyura Bythograeidae Austinograea alayseae Guinot, 1990 JQ035660
Austinograea alayseae KC851803
Austinograea rodriguezensis Tsuchida & Hashimoto, 2002 JQ035658
Gandalfus puia McLay, 2007 KR002727
Pleocyemata Brachyura Bythograeidae Gandalfus yunohana (Takeda, Hashimoto & Ohta, 2000) EU647222
Segonzacia mesatlantica (Williams, 1988) KY541839
Dotillidae Ilyoplax deschampsi (Rathbun, 1913) JF909979
Dynomenidae Dynomene pilumnoides Alcock, 1900 KT182070
Eriphiidae Pseudocarcinus gigas (Lamarck, 1818) AY562127
Geryonidae Chaceon sp. BZ-2016 KU507298
Grapsidae Pachygrapsus crassipes (Randall, 1840) KC878511
Homolidae Homola orientalis Henderson, 1888 KT182071
Homologenus malayensis Ihle, 1912 KJ612407
Moloha majora (Kubo, 1936) KT182069
Leucosiidae Pyrhila pisum (De Haan, 1841) KU343210
Macrophthalmidae Macrophthalmus japonicus (De Haan, 1835) KU343211
Majidae Maja crispata Risso, 1827 KY650651
Maja squinado (Herbst, 1788) KY650652
Matutidae Ashtoret lunaris (Forskål, 1775) LK391941
Menippidae Myomenippe fornasinii (Bianconi, 1851) LK391943
Mictyridae Mictyris longicarpus Latreille, 1806 LN611670
Mithracidae Damithrax spinosissimus (Lamarck, 1818) KM405516
Ocypodidae Tubuca arcuata (De Haan, 1835) KX911977
Ocypode ceratophthalmus (Pallas, 1772) LN611669
Ocypode cordimanus Latreille, 1818 KT896743
Oregoniidae Chionoecetes japonicus pacificus Sakai, 1978 AB735678
Parathelphusidae Somanniathelphusa boyangensis Dai, Peng & Zhou, 1994 KU042042
Portunidae Callinectes sapidus Rathbun, 1896 AY363392
Charybdis feriata (Linnaeus, 1758) KF386147
Charybdis japonica (A. Milne-Edwards, 1861) FJ460517
Charybdis natator (Herbst, 1794) MF285241
Portunus pelagicus (Linnaeus, 1758) KM977882
Portunus pelagicus KR153996
Portunus pelagicus KT382858
Portunus sanguinolentus (Herbst, 1783) KT438509
Portunus trituberculatus (Miers, 1876) AB093006
Scylla olivacea (Herbst, 1796) FJ827760
Scylla paramamosain Estampador, 1949 FJ827761
Scylla paramamosain JX457150
Scylla serrata (Forskål, 1775) FJ827758
Scylla serrata HM590866
Scylla tranquebarica (Fabricius, 1798) FJ827759
Thalamita crenata Rüppel, 1830 LK391945
Potamidae Geothelphusa dehaani (White, 1847) AB187570
Huananpotamon lichuanense Dai, Zhou & Peng, 1995 KX639824
Longpotamon xiushuiense (Dai, Zhou & Peng, 1995) KU042041
Potamiscus motuoensis Dai, 1990 KY285013
Raninidae Lyreidus brevifrons Sakai, 1937 KM983394
Ranina ranina (Linnaeus, 1758) KM189817
Umalia orientalis (Sakai, 1963) KM365084
Sesarmidae Clistocoeloma sinense Shen, 1933 KU589292
Metopaulias depressus Rathbun, 1896 KX118277
Parasesarma tripectinis (Shen, 1940) KU343209
Sesarma neglectum de Man, 1887 KX156954
Sesarmops sinensis (H. Milne Edwards, 1853) KR336554
Varunidae Cyclograpsus granulosus H. Milne Edwards, 1853 LN624373
Eriocheir sinensis H. Milne Edwards, 1853 AY274302
Eriocheir sinensis KM516908
Eriocheir sinensis KP064329
Eriocheir sinensis KP126617
Eriocheir sinensis KY041629
Helicana wuana (Rathbun, 1931) KX344898
Helice latimera Parisi, 1918 KU589291
Helice tientsinensis Rathbun, 1931 KR336555
Hemigrapsus sanguineus (De Haan, 1835) KX456205
Xanthidae Leptodius sanguineus (H. Milne Edwards, 1834) KT896744
Xenograpsidae Xenograpsus ngatama McLay, 2007 KY985236
Xenograpsus testudinatus Ng, Huang & Ho, 2000 EU727203

The new universal primers for decapod eDNA were designed on the 16S rRNA gene (for details, see Results and Discussion) and were named MiDeca-F/R (F and R represent forward and reverse, respectively).

In silico evaluation of variation in the target region

Interspecific differences within the amplified DNA sequences are required for accurate taxonomic assignments. To computationally evaluate levels of interspecific variations within the target region (hereafter called “MiDeca sequence”) across different taxonomic groups of decapods, 254 whole mitogenome sequences used for the primer development were subjected to calculation of pairwise edit distances (intra-species, inter-species, inter-genus and inter-family levels, respectively). The edit distance is defined as the minimum number of single-nucleotide substitutions, insertions or deletions that are required to transform one sequence into the other. (Jones and Pevzner 2004).

Test of primers with tissue extracted DNA

In order to examine the performance of MiDeca primers, extracted DNA from a single individual of each 250 species across the suborder Dendrobranchiata and 10 of 11 infraorders of the suborder Pleocyemata was used to amplify MiDeca sequences (Table 2). Total genomic DNA was extracted from each tissue (thoracic or pleon muscle or pereopod or pleopod muscle), which was preserved in 70–99% ethanol for one to more than 10 years, using a DNeasy Blood & Tissue kit (Qiagen, Hilden, Germany) with an elution volume of 100 µl.

A list of decapod species for testing MiDeca primers (without adapter sequences) using extracted DNA subsequently sequenced with a Sanger method. Institutional abbreviation: AMF, Aquamarine Fukushima, Iwaki, Japan.

Suborder/Infraorder Family Species CBM reg. No. Acc. No. bp
Dendrobranchiata Penaeidae Metapenaeopsis dalei (Rathbun, 1902) 15064 LC464519 168
Metapenaeus brevicornis (H. Milne Edwards, 1837) 13761 LC430751 170
Penaeus latisulcatus Kishinouye, 1896 14890 LC430718 168
Trachysalambria curvirostris (Stimpson, 1860) 14210 LC430766 169
Solenoceridae Haliporoides sibogae (de Man, 1907) 14982 LC430749 167
Pleoticus muelleri (Spence Bate, 1888) 14981 LC430764 167
Sicyonidae Sicyonia laevis Spence Bate, 1881 14851 LC464520 167
Sicyonia truncata (Kubo, 1949) 15110 LC464521 166
Sergestidae Sergia talismani (Barnard, 1947) 14966 LC430728 172
Sicyonella antennata Hansen, 1919 14897 LC430716 165
Pleocyemata/Stenopodidea Stenopodidae Richardina parvioculata Saito & Komatsu, 2009 15048 LC430808 158
Pleocyemata/Caridea Pasiphaeidae Leptochela robusta Stimpson, 1860 14948 LC464522 160
Leptochela sydniensis Dakin & Colefax, 1940 14965 LC430727 161
Atyidae Neocaridina davidi (Bouvier, 1904) 14978 LC430762 163
Disciadidae Discias exul Kemp, 1922 15285 LC469628 150
Palaemonidae Macrobrachium nipponense (De Haan, 1849) 14986 LC430769 159
Palaemon debilis Dana, 1852 14835 LC464524 162
Palaemon macrodactylus Rathbun, 1902 14968 LC464526 162
Palaemon pacificus (Stimpson, 1860) 15087 LC464525 160
Palaemon paucidens De Haan, 1844 14979 LC430763 162
Palaemon serrifer (Stimpson, 1860) 15298 LC464527 160
Palaemon orientis Holthuis, 1950 13697 LC430771 160
Phycomenes indicus (Kemp, 1915) 14899 LC464528 159
Cuapetes grandis (Stimpson, 1860) 14768 LC464523 160
Alpheidae Alpheus hailstonei Coutière, 1905 14949 LC464536 157
Alpheus pacificus Dana, 1852 14688 LC464538 160
Alpheus heeia Banner & Banner, 1975 14762 LC464537 159
Alpheus ikedosoma Komai, 2015 14825 LC430798 158
Alpheus longipalma Komai & Ohtomi, 2018 14484 LC430730 159
Alpheus sp. aff. pacificus 15299 LC472887 160
Arete dorsalis (Stimpson, 1860) 14756 LC464539 155
Athanas japonicus Kubo, 1936 14658 LC464540 159
Betaeus granulimanus (Yokoya, 1927) 14240 LC464541 158
Caligoneus cavernicola Komai & Fujita, 2018 14136 LC464542 183
Salmoneus brucei Komai, 2009 14558 LC464543 158
Synalpheus tumidomanus (Paulson, 1875) 15089 LC464544 158
Ogyrididae Ogyrides orientalis (Stimpson, 1860) 14824 LC430799 154
Hippolytidae Latreutes acicularis (Ortmann, 1890) 14682 LC464529 155
Latreutes porcinus Kemp, 1916 14900 LC430717 163
Saron marmoratus (Olivier, 1811) 14911 LC430713 171
Thoridae Eualus biungius (Rathbun, 1902) 15178 LC430743 161
Eualus kikuchii Miyake & Hayashi, 1967 14516 LC430740 162
Eualus leptognathus (Stimpson, 1860) 14503 LC464530 160
Heptacarpus acuticarinatus Komai & Ivanov, 2008 14507 LC430737 161
Heptacarpus futilirostris (Spence Bate, 1888) 14056 LC464531 161
Heptacarpus geniculatus (Stimpson, 1860) 14055 LC464532 161
Heptacarpus jordani (Rathbun, 1902) 14993 LC464533 163
Lebbeus armatus (Owen, 1839) 14316 LC464534 161
Lebbeus kuboi Hayashi, 1992 15177 LC430742 161
Thor paschalis (Heller, 1862) 14973 LC430702 161
Spirontocaris prionota (Stimpson, 1864) 14058 LC464535 162
Pandalidae Heterocarpus hayashii Crosnier, 1988 14983 LC430767 163
Pandalopsis glabra Kobjakova, 1936 AMF-ZC 0012 LC430745 160
Pandalopsis miyakei Hayashi, 1986 8898 LC430744 160
Plesionika semilaevis Spence Bate, 1888 14984 LC430768 159
Glyphocrangonidae Glyphocrangon formosana Komai, 2004 15054 LC469627 165
Crangonidae Aegaeon lacazei (Gourret, 1887) 14860 LC464545 159
Crangon uritai Hayashi & Kim, 1999 15084 LC464546 164
Parapontophilus demani (Chace, 1983) 15038 LC430760 161
Philocheras coralliophilus Komai & Kim, 2010 15027 LC430747 165
Philocheras japonicus (Doflein, 1902) 15045 LC430761 159
Philocheras parvirosotris (Kemp, 1916) 14831 LC430800 155
Syncrangon angusticauda (De Haan, 1849) 14212 LC472888 160
Pleocyemata/Astacidea Nephropidae Nephropsis stewarti Wood-Mason, 1872 14212 LC430805 163
Pleocyemata/Axiidea Axiidae Eiconaxius farreae Ortmann, 1890 14870 LC464548 158
Litoraxius boshu Komai & Tachikawa, 2007 9225 LC472889 162
Strahlaxiidae Neaxius acanthus (A. Milne-Edwards, 1878) 14990 LC430806 165
Callianassidae Cheramus spinicauda Komai, Maenosono & Fujita, 2014 15139 LC464550 159
Neocallichirus jousseaumei (Nobili, 1904) 15026 LC430756 161
Nihonotrypaea harmandi (Bouvier, 1903) 14728 LC430796 160
Nihonotrypaea japonica (Ortmann, 1890) 15176 LC430729 160
Nihonotrypaea petalura (Stimpson, 1860) 15003 LC464551 162
Paratrypaea bouvieri (Nobili, 1904) 14812 LC464552 159
Eucalliacidae Calliaxina novaebritanniae (Borradaile, 1900) 14653 LC464549 162
Eucalliax aequimana (Baker, 1904) 15028 LC430748 160
Pleocyemata/Gebiidea Laomediidae Laomedia astacina De Haan,1849 13759 LC430775 163
Upogebiidae Tuerkayogebia kiiensis (Sakai, 1971) 15218 LC472890 176
Pleocyemata/Achelata Palinuridae Panulirus japonicus (von Siebold, 1824) 14991 LC430807 171
Pleocyemata/Polychelida Polychelidae Stereomastis helleri (Spence Bate, 1878) 10076 LC430804 172
Pleocyemata/Anomura Galatheidae Coralliogalathea humilis (Nobili, 1905) 14896 LC430703 163
Galathea orientalis Stimpson, 1858 15014 LC430817 163
Munididae Munida multilineata Komai, 2012 14494 LC430735 163
Munida munin Komai, 2011 15154 LC464553 166
Raymunida elegantissima (de Man, 1902) 14939 LC464554 164
Munidopsidae Munidopsis petalorhyncha Baba, 2005 13528 LC430765 164
Porcellanidae Novorostrum decorocrus Osawa, 1998 14801 LC464555 166
Pachycheles hertwigi Balss, 1913 15015 LC430818 161
Pachycheles stevensii Stimpson, 1858 15016 LC430813 161
Petrolisthes asiaticus Leach, 1820 14912 LC430712 167
Petrolisthes japonicus (De Haan, 1849) 15017 LC430816 166
Pisidia dispar Stimpson, 1858 14758 LC464556 163
Polyonyx sinensis Stimpson, 1858 13700 LC430772 165
Polyonyx utinomii Miyake, 1943 14360 LC464557 165
Lithodidae Paralomis verrilli Benedict, 1894) 15179 LC430803 169
Diogenidae Aniculus miyakei Forest, 1984 14628 LC430788 167
Calcinus vachoni Forest, 1954 15009 LC430811 165
Clibanarius virescens (Krauss, 1843) 15008 LC464558 170
Dardanus pedunculatus (Herbst, 1804) 14629 LC430754 165
Diogenes avarus Heller, 1865 14555 LC464559 165
Diogenes spinifrons (De Haan, 1849) 14828 LC430797 167
Paguristes albimaculatus Komai, 2001 14852 LC464560 170
Paguristes ortmanni Miyake, 1978 15246 LC464561 169
Pseudopaguristes laurentae Morgan & Forest, 1991 14853 LC430725 189
Paguridae Alainopaguroides andamanensis McLaughlin, 2000 14954 LC464562 167
Alloeopagurodes spiniacicula Komai, 1998 15173 LC430734 167
Catapaguroides japonicus de Saint Laurent, 1968 15300 LC464563 169
Decaphyllus spinicornis de Saint Laurent, 1968 14863 LC464564 165
Pagurixus anceps (Forest, 1954) 14565 LC464565 167
Pagurus filholi (de Man, 1887) 15161 LC464566 168
Pagurus imafukui McLaughlin & Konishi, 1994 14862 LC464567 167
Pagurus japonicus (Stimpson, 1858) 15011 LC464568 168
Pagurus maculosus Komai & Imafuku, 1996 15012 LC464569 168
Pagurus nigrivittatus Komai, 2003 15013 LC430810 168
Parapaguridae Parapagurus furici Lemaitre, 1999 15037 LC430757 172
Pleocyemata/Brachyura Dromiidae Cryptodromia bullifera (Alcock, 1900) 14249 LC469629 161
Metadromia wilsoni (Fulton & Grant, 1902) 14251 LC469630 161
Dynomenidae Dynomene pilumnoides Alcock, 1900 14951 LC469631 164
Cyclodorippidae Tymolus japonicus Stimpson, 1858 14864 LC469632 152
Cymonomidae Cymonomus curvirostris Sakai, 1963 14849 LC469633 167
Homolidae Homolochunia gadaletae Guinot & Richer de Forges, 1995 15039 LC469634 167
Latreillopsis bispinosa Henderson, 1888 15033 LC430759 167
Yaldwynopsis saguili Richer de Forges & Ng, 2007 14252 LC430792 169
Calappidae Calappa gallus (Herbst, 1803) 14630 LC430784 171
Calappa hapatica (Linnaeus, 1758) 15006 LC469635 170
Mursia orientalia Takeda & Galil, 2005 14513 LC430739 167
Matutidae Ashtoret miersi (Henderson, 1887) 14989 LC430704 159
Izanami curtispina (Sakai, 1961) 15153 LC469636 159
Pleocyemata/Brachyura Ethusidae Ethusa quadrata Sakai, 1937 14492 LC430733 164
Leucosiidae Arcania cornigera Naruse, 2014 14857 LC469637 159
Ebalia tuberculosa (A. Milne-Edwards, 1873) 14850 LC469638 160
Merocryptus lambryformis A. Milne-Edwards, 1873 14856 LC469639 159
Nucia speciosa Dana, 1852 14261 LC469640 158
Philyra kanekoi Sakai, 1934 14967 LC430795 160
Philyra syndactyla Ortmann, 1892 15146 LC469641 163
Pseudophilyra intermedia Ihle, 1918 14379 LC469642 161
Tokoyo eburnea (Alcock, 1896) 14866 LC469643 160
Inachidae Cyrtomaia owstoni Terazaki, 1903 15040 LC469644 164
Epialtidae Huenia heraldica De Haan, 1839 14632 LC430776 165
Menaetius monoceros (Latreille, 1825) 14778 LC430721 148
Naxioides taurus (Poock, 1890) 15152 LC469645 163
Oxypleurodon stimpsoni (Miers, 1886) 15118 LC469646 161
Phalangipus hystrix (Miers, 1886) 15080 LC469647 164
Pugettia minor Ortmann, 1893 14855 LC469648 169
Pugettia nipponensis Rathbun, 1932 15035 LC469649 170
Pugettia quadridens (De Haan, 1839) 15018 LC430812 170
Majidae Choniognathus reini (Balss, 1924) 14867 LC469650 161
Micippa philyra (Herbst, 1803) 15019 LC469651 159
Paramaia spinigera (De Haan, 1837) 14636 LC430786 169
Prismatopus harmandi (Bouvier, 1906) 14633 LC469651 174
Schizophrys aspera (H. Milne Edwards, 14637 LC430777 166
Oregoniidae Pleistacantha sanctijohannis Miers, 1879) 14495 LC430736 164
Pleisticanthoides simplex (Rathbun, 1932) 15116 LC469653 172
Cancridae Glebocarcinus amphioetus (Rathbun, 1898) 14988 LC430701 177
Romaleon gibbosulum (De Haan, 1833) 14858 LC469654 174
Parthenopidae Rhinolambrus contrarius (Herbst, 1804) 14639 LC430779 165
Aethridae Aethra edentata Edmondson, 1951 14264 LC469655 168
Pilumnidae Actumnus setifer (De Haan, 1835) 14382 LC469656 156
Actumnus squamosus (De Haan, 1835) 14648 LC430781 156
Actumnus uformis Takeda & Komatsu, 2017 14944 LC469657 161
Benthopanope pearsei (Rathbun, 1932) 14918 LC430707 168
Calmania balssi (Sakai, 1935) 14869 LC469658 162
Cryptolutea sagamiensis (Sakai, 1935) 14512 LC430738 160
Harrovia japonica Balss, 1921 14312 LC469659 164
Heteropilumnus ciliatus (Stimpson, 1858) 11291 LC469660 177
Pilumnus longicornis Hilgendorf, 1878 11133 LC469661 165
Pilumnus minutus De Haan, 1835 14335 LC469662 165
Pilumnus tomentosus Latreille, 1825 14310 LC430794 164
Pilumnus sp.1 14941 LC469663 165
Pseudolitochira integra (Miers, 1884) 14917 LC430706 160
Typhlocarcinus sp. 14522 LC469664 161
Zehntneriana tadafumii Lee, Kim & Ng, 2015 14916 LC430705 162
Xenophthalmodes morsei Rathbun, 1932 14961 LC430724 162
Portunidae Charybdis acuta (A. Milne-Edwards, 1869) 14647 LC430785 168
Charybdis natator (Herbst, 1794) 14640 LC430783 168
Carupa ohashii Takeda, 1983 14269 LC469665 173
Cycloachelous granulatus (H. Milne Edwards, 1834) 15095 LC469666 174
Laleonectes nipponensis (Sakai, 1938) 14272 LC469667 167
Portunus pelagicus (Linnaeus, 1758) 15168 LC469668 170
Scylla olivacea (Herbst, 1796) 15169 LC469669 169
Thalamita chaptali (Audoiun, 1826) 15200 LC469670 167
Thalamita longifrons (A. Milne-Edwards, 1869) 15158 LC469673 167
Thalamita picta Stimpson, 1858 12359 LC469671 167
Thalamita sima H. Milne Edwards, 1834 14646 LC430780 164
Thalamita seurati Nobili, 1906 12692 LC469672 167
Thalamita sp. aff. coeruleipes 15197 LC469674 165
Thranita pelsarti (Mondgomery, 1931) 14650 LC430787 164
Xiphonectes brocki (de Man, 1887) 14905 LC430719 172
Xiphonectes macrophthalmus (Rathbun, 1906) 14276 LC469675 171
Geothelphusa dehaani (White, 1847) 14231 LC430731 169
Goneplacidae Notonyx angulatus Naruse & Takeda, 2010 14314 LC469676 167
Pedroplax megalops (Takeda, 1977) 14950 LC469677 174
Pycnoplax surugensis (Rathbun, 1932) 15036 LC430758 170
Xanthidae Actaea bocki Odhner, 1925 14856 LC469678 168
Actaea semblatae Guinot, 1976 14288 LC469679 171
Pleocyemata/Brachyura Xanthidae Alainodaeus nuku Davie, 1997 14942 LC469680 170
Atergatis floridus (Linnaeus, 1767) 14641 LC430782 170
Atergatis granulatus de Man, 1889 14285 LC430793 170
Atergatis integerrimus (Lamarck, 1818) 14286 LC469681 171
Atergatis subdentatus (De Haan, 1835) 14642 LC430789 172
Banareia subglobosa (Stimpson, 1858) 14644 LC430790 169
Cycloxanthops truncatus (De Haan, 1837) 15021 LC430809 169
Danielea noelensis (Ward, 1942) 14296 LC469682 169
Euxanthus herdmani Laurie, 1906 14292 LC469683 167
Etisus electra (Herbst, 1801) 14974 LC430710 171
Etisus frontalis (Dana, 1852) 14975 LC430711 171
Etisus laevimanus Randall, 1840 14643 LC430791 170
Forestiana granulata (Krauss, 1843) 15155 LC469684 171
Gaillardiellus orientalis (Odhner, 1925) 8363 LC469685 169
Lachnopodus subacutus (Stimpson, 1858) 14913 LC430708 168
Leptodius affinis (De Haan, 1835) 15022 LC430815 167
Leptodius sanguineus (H. Milne Edwards, 1834) 14914 LC430709 169
Lophozozymus dodone (Herbst, 1801) 14283 LC469686 170
Liomera caelata (Odhner, 1925) 15172 LC469687 169
Liomera rugata (H. Milne Edwards, 1834) 14907 LC430715 169
Medaeops granulosus (Haswell, 1882) 13757 LC430752 171
Metaxanthops acutus Serène, 1984 14308 LC469688 168
Microcassiope orientalis Takeda & Miyake, 1969 14301 LC469689 169
Neoliomera pubescens (H. Milne Edwards, 1834) 14282 LC469690 175
Palapedia rastripes (Müller, 1887) 14906 LC430714 168
Palapedia truncatifrons (Sakai, 1974) 13758 LC430753 168
Paraxanthias elegans (Stimpson, 1858) 15023 LC430814 170
Pilodius areolatus (Milne Edwards, 1834) 14290 LC469691 168
Platypodia granulosa (Rüppel, 1830) 14959 LC430722 171
Eriphiidae Eriphia scabricula Dana, 1852 15143 LC469692 166
Oziidae Epixanthus corrosus A. Milne-Edwards, 1873 14772 LC430720 173
Grapsidae Pachygrapsus minutus (A. Milne Edwards, 1873) 15144 LC430755 171
Sesarmidae Clistocoeloma sinense Shen, 1933 14664 LC469693 179
Nanosesarma minutum (de Man, 1887) 15166 LC469694 175
Parasesarma affine (De Haan, 1837) 14992 LC469695 183
Parasesarma pictum (De Haan, 1835) 14838 LC469696 184
Plagusiidae Guinusia dentipes (De Haan, 1835) 14645 LC430778 169
Percnidae Percnon planissimum (Herbst, 1804) 14571 LC469697 176
Varunidae Acmaeopleura parvula Stimpson, 1848 14848 LC469698 171
Chasmagnathus convexus (De Haan, 1835) 14662 LC469699 171
Cyclograpsus intermedius Ortmann, 1894 14847 LC469700 173
Gaetice depressus (De Haan, 1835) 15024 LC469701 169
Gopkittisak angustum Komai, 2011 14561 LC469702 165
Helice tridens (De Haan, 1835) 13695 LC430770 172
Hemigrapsus longitarsis (Miers, 1879) 15174 LC469703 174
Hemigrapsus takanoi Asakura & Watanabe, 2005 13696 LC430750 175
Ptychognathus barbatus (A. Milne-Edwards, 1873) 15029 LC430746 172
Sestrostoma depressum (Sakai, 1965) 15175 LC469704 172
Camptandriidae Deiratonotus cristatum (de Man, 1895) 13760 LC430774 171
Macrophthalmidae Macrophthalmus brevis (Herbst, 1804) 15201 LC469705 172
Macrophthalmus convexus Stimpson, 1858 14841 LC469706 172
Macrophthalmus serenei Takeda & Komai, 1991 14556 LC469707 174
Macrophthalmus telescopicus (Owen, 1839) 14384 LC469708 171
Tritodynamia japonica Ortmann, 1894 14228 LC430732 171
Pinnotheridae Pinnotheres pholadis De Haan, 1835 14225 LC430773 171
Hexapodidae Hexapinus simplex Rahayu & Ng, 2014 14393 LC430741 168

DNA concentrations were measured and recorded with a NanoDrop Lite spectrophotometer (Thermo Fisher Scientific, DE, USA). PCR was carried out with 30 cycles of a 8.0 µl reaction volume (divided from the original PCR mastermix) containing 2.2 µl sterile distilled water, 3.8 µl 2 × Gflex PCR buffer (Takara, Otsu, Japan), 0.4 µl of each primer (5 µM), 0.2 µl Tks Gflex DNA polymerase (Takara, Otsu, Japan) and 1.0 µl template. The thermal cycle profile after an initial 1 min denaturation at 94 °C was as follows: denaturation at 98 °C for 10 s, annealing at 50 °C for 10 s and extension at 68 °C for 10 s with the final extension at the same temperature for 7 min. The PCR products were subjected to agarose gel electrophoresis using 2% L03 (Takara, Otsu, Japan) to confirm the amplifications. The PCR products were purified using Exo SAP-IT (USB, OH, USA) to remove redundant dNTPs and primers.

Direct sequencing of the purified PCR products was performed with the ABI 3130xl Genetic Analyzer (Life Technologies, CA, USA) and dye-labelled terminators (BigDye terminator v. 1.1; Applied Biosystems, CA, USA) following the manufacture’s protocol. The DNA sequences were edited and assembled by GENETYX-MAC v. 17 (Genetyx, Tokyo, Japan) or MEGA7 and registered in the DDBJ/EMBL/NCBI database (Table 2).

Water sampling and filtration

In order to test the versatility of the newly designed primers (MiDeca-F/R), we used a filtered seawater sample collected at the rocky shore of Banda, Tateyama City, Chiba Prefecture (34.9758N, 139.7675E) on 14 September 2017 (Figs 1, 2), where the decapod crustacean fauna is well documented.

Figure 1.

Map of Japan, showing the location of the sea water sampling site (Banda, Tateyama, Chiba Prefecture). This map is based on the aerial photography published by Geospatial Information Authority of Japan.

Figure 2.

A, sampling site at Banda, Tateyama, Chiba Prefecture; B, sea water sampling operation with a bucket.

An on-site filtration method was employed to collect decapod eDNA. Disposable gloves were worn and changed between each sample. The sampling equipment (8 litre polyethylene bucket) was thoroughly decontaminated with a 10% bleach solution before use. Surface water was collected using 10 casts of the bucket fastened to a 15 m rope. In each cast, approximately 50 ml seawater was drawn into a disposable syringe with the lure lock connector (50 ml; TERUMO, Tokyo, Japan), an inlet port of the 0.45 µm Sterivex filter cartridge (Merck Millipore, MA, USA) was attached to the syringe and the seawater was filtered on to the membrane by pushing the plunger. This step was repeated twice in a single cast and the final filtration volume reached 1000 ml with 10 casts of the bucket.

After the on-site filtration, an outlet port of the filter cartridge was sealed with the Parafilm (LMS, Tokyo, Japan), RNAlater (1.6 ml; Thermo Fisher Scientific, DE, USA) was added into the cartridge using a disposable pipette to prevent eDNA degradation and an inlet port was sealed with the film. A filtration blank was made by filtering 500 ml of pure water in the same manner at the end of the water sampling. Filter cartridges were transported to the laboratory in a cooler with ice packs and then kept at -20 °C in the freezer prior to eDNA extractions.

eDNA was extracted from Sterivex cartridges using a DNeasy Blood & Tissue kit (Qiagen) following the method developed by Miya et al. (2016) with slight modifications. An inlet port of each Sterivex cartridge was connected with a 2.0 ml collection tube and the connection between the cartridge and collection tube was tightly sealed with the Parafilm. The combined unit was inserted into a centrifuge adaptor for a 15 ml conical tube and was centrifuged at 6000g for 1 min to remove seawater and RNAlater for DNA extraction. In order to completely remove liquid remaining in the cartridge, an aspirator (QIAvac 24 Plus, Qiagen) was used. The Sterivex cartridge was subjected to lysis using proteinase K. Before the lysis, PBS (220 μl), proteinase K (20 μl) and buffer AL (200 μl) were mixed and the mixed solution was gently pipetted into the Sterivex cartridge from an inlet port. The Sterivex cartridge was again sealed and then incubated in a 56 °C preheated incubator for 20 min, using a rotator (Mini Rotator ACR-100, As One) with a rotation rate of 10 rpm. After the incubation, the Sterivex cartridge connected with a 2 ml tube (DNA LowBind tube, SARSTEDT), which was placed in a 50 ml conical tube, was centrifuged at 6000g for 1 min to collect the DNA. The collected DNA solution (ca. 900 μl) was purified using the DNeasy Blood and Tissue kit following the manufacture’s protocol.

Library preparation and MiSeq sequencing with eDNA sample

eDNA extracted from the seawater sample collected at Banda was subjected to the first-round PCR (1st PCR) and the second-round PCR (2nd PCR) in order to append amplified sequences with three kinds of adaptor sequences: 1) primer-binding sites for sequencing; 2) dual-index sequences to distinguish amplicons; and 3) sequences for binding to the flowcells of the Illumina MiSeq (Illumina, CA, USA).

The 1st PCR was carried out with 38 cycles of a 12 µl reaction volume containing 6.0 µl 2 × KAPA HiFi HotStart ReadyMix (KAPA Biosystems, MA, USA), 1.4 µl of each MiDeca primer (5 µM primer F/R), 1.2 µl sterile distilled H2O and 2.0 µl eDNA template. In order to minimise PCR dropouts, 8 replications were performed for the 1st PCR using a 0.2 ml 8-strips tube.

The thermal cycle profile after an initial 3 min denaturation at 95 °C was as follows: denaturation at 98 °C for 20 s, annealing at 60 °C for 15 s and extension at 72 °C for 15 s with the final extension at the same temperature for 5 min. The 1st PCR products from the 8 tubes were pooled in a single 1.5 ml tube and the pooled products were purified using a GeneRead Size Selection kit (Qiagen, Hilden, Germany) in order to remove dimers and monomers following the manufacturer’s protocol. Subsequently, the purified products were quantified using TapeStation 2200 (Agilent, Tokyo, Japan), diluted to 0.1 ng/µl using Milli Q water and the diluted products were used as a template for the 2nd PCR.

The 2nd PCR was conducted with 12 cycles of a 15 µl reaction volume containing 7.5 µl 2 × KAPA HiFi HotStart ReadyMix, 0.9 µl each primer (5 µM), 3.9 µl sterile distilled H2O and 1.9 µl template. The thermal cycle profile after an initial 3 min denaturation at 95 °C was as follows: denaturation at 98 °C for 20 s, annealing and extension combined at 72 °C (shuttle PCR) for 15 s with the final extension at the same temperature for 5 min.

In order to monitor contamination during the process of PCRs, blank samples were prepared. During the 1st PCR, a filtration blank (FB), an extraction blank (EB) and a PCR blank (1B) with 2.0 µl milli Q water instead of template eDNA were added; during the 2nd PCR, in addition to the three blanks used during the 1st PCR, one more PCR blank (2B) was added.

All the libraries containing the target region and the three adapter sequences were mixed in equal volume and the pooled libraries were size-selected from approximately 340 bp using a 2% E-Gel Size Select agarose gel (Invitrogen, CA, USA). The concentration of the size-selected libraries was measured using a Qubit dsDNA HS assay kit and a Qubit fluorometer (Life Technologies, CA, USA) and sequenced on the MiSeq platform using a MiSeq v2 Reagent Kit for 2 × 150 bp PE (Illumina, CA, USA) following the manufacturer’s protocol.

Data preprocessing and taxonomic assignment

All data preprocessing and analysis of MiSeq raw reads were performed using USEARCH v10.0.240 (Edgar 2010) according to the following steps.

1) Both forward and reverse reads were merged by aligning them using the fastq_mergepairs command. During this process, low-quality tail reads with a cut-off threshold set at a quality (Phred) score of 2, too short reads (< 64 bp) after tail trimming and those paired reads with too many differences (> 5 positions) in the aligned region (ca. 70 bp) were discarded; 2) primer sequences were removed from those merged reads using the fastx_truncate command; 3) those reads without the primer sequences underwent quality filtering using the fastq_filter command to remove low quality reads with an expected error rate of > 1% and too short reads of < 50 bp; 4) the preprocessed reads were dereplicated using the fastx_uniques command and all singletons, doubletons and tripletons were removed from the subsequent analysis following the recommendation by Edgar (2010); 5) the dereplicated reads were denoised using the unoise3 command and all putatively chimeric and erroneous sequences were separated from the subsequent OTU assignment; 6) finally all processed reads were assigned to OTU with a sequence identity of > 98% (query coverage ≥ 90%, 2 or 3 nucleotide differences allowed) using the usearch_global command. Reads with a sequence identity of 80–98% were also assigned to “U98 OTU” and were subjected to clustering at the level of 0.98 using cluster_smallmem command. All of these outputs were tabulated with read abundances.

For reference sequences, decapod crustacean mitochondrial 16S rRNA gene sequences were downloaded from NCBI as of 23 June 2018 and MiDeca sequences were extracted using custom Perl scripts and used as the reference database during taxonomic assignment. In addition to those published sequences, we independently determined MiDeca sequences from 250 decapod crustaceans (Table 2) and added those sequences to the reference database (27,236 reference sequences in total as of 26 September 2018; those sequences are represented by 4005 decapod taxa identified to species from 1135 genera of 167 families of 11 infraorders/suborders).

Results

MiDeca primers

By visual inspection of the two aligned mitochondrial rRNA genes from 254 sequences, two conservative regions (ca. 20 bp) that flank a hyper-variable region (154–184 bp with the exception of one extremely short sequence of 68 bp for a sergestid Acetes chinensis) were identified within the 16S rRNA gene. The new PCR primers for metabarcoding eDNA from decapods were designed on the basis of these two conservative regions and the primers were named MiDeca. The MiDeca-forward primer (MiDeca-F) comprised 5´-GGA CGA TAA GAC CCT ATA AA-3´ (20 mer), whereas the MiDeca-reverse primer (MiDeca-R) comprised 5´-ACG CTG TTA TCC CTA AAG T-3´ (19 mer). Melting temperatures and G/C contents of the MiDeca-F/R primers are 51.1 °C/51.6 °C and 40.0%/42.1%, respectively. Nucleotide variation in the primer region amongst the 207 species used to design MiDeca is summarised in Table 3.

Nucleotide sequences of the universal primers (MiDeca) and base compositions in the selected 254 sequences (see Table 1). This forward (F) and reversal (R) primer pair amplifies the mid region of the mitochondrial 16S rRNA gene with a mean length of 164 bp (154–184 bp; except for one unusually short sequence from Acetes chinensis).

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
MiDeca-F 5′ G G A C G A T A A G A C C C T A T A A A 3′
A 20 0 254 2 2 254 1 253 254 0 254 0 1 0 0 236 0 236 254 254
T 4 0 0 1 0 0 238 1 0 0 0 0 0 0 254 0 252 0 0 0
G 230 254 0 0 252 0 0 0 0 254 0 0 0 0 0 18 1 18 0 0
C 0 0 0 251 0 0 15 0 0 0 0 254 253 254 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0
MiDeca-R 3′ T G A A A T C C C T A T T G T C G C A 5′
A 254 0 0 0 0 254 0 0 0 253 0 254 254 0 254 0 0 3 0
T 0 0 254 237 237 0 0 0 0 1 254 0 0 0 0 0 3 0 254
G 0 0 0 0 0 0 254 254 254 0 0 0 0 0 0 254 0 251 0
C 0 254 0 17 17 0 0 0 0 0 0 0 0 254 0 0 251 0 0

With the use of tissue extracted DNA, MiDeca primers, without adapter sequences, were able to amplify each variable region of 250 decapod species from 10 suborders/infraorders (Table 2) and those nucleotide sequences were determined using the Sanger method. The lengths of MiDeca sequences vary from 148 bp to 189 bp (mean 165.7 bp). Those sequences were deposited in DDBJ/EMBL/GenBank databases (Table 2).

In silico evaluation of variation in MiDeca sequence

The pairwise edit distances from MiDeca sequences were calculated for 254 sequences distributed across 10 infraorders, 56 families, 123 genera and 207 species and the results were sorted into between-families, genus, species and within species (Fig. 3). Each median of the edit distance was 41, 47, 35 and 4, respectively.

Figure 3.

Summary of intraspecific, inter-species, inter-genus and inter-family edit distances of MiDeca sequences from 254 decapods used for the primer development.

eDNA detection from natural seawater

In total, the MiSeq paired-end sequencing yielded a total of 4,693,875 raw reads with an average of 95.2% base calls being Phred quality scores of more than or equal to 30.0 (Q30; error rate = 0.1% or base call accuracy = 99.9%). This run was highly successful considering that the quality scores specified by Illumina are more than 80% bases higher than Q30 at 2 × 150 bp (Illumina Publication no. 770-2011-001 as of 27 May 2014).

Of the 4,693,875 raw reads, our sample from Tateyama Bay comprised 185,690 raw reads and they were merged, quality-filtered, dereplicated and denoised, resulting in a total of 161,753 reads (87.1% raw reads being retained). Preprocessed reads from the four blanks (FB, EB, 1B, 2B) were minor, comprising only 7–128 reads (0.004–0.07% of the non-negative sample). We therefore considered that those reads from the four blanks were negligible and they were not used in the subsequent taxonomic assignment.

The preprocessed reads from Tateyama Bay sample were subjected to taxonomic assignment with the custom database. Finally, these reads were assigned to 35 crustacean species with the sequence identity of 98–100%, 10 crustacean species with the sequence identity of 80–98% (Table 4) and 69 no-hit taxa. Of the 35 species with the identity of 98–100%, 34 were decapods with the exception of the amphipod Caprella scaura. Of those 34 decapod species, the occurrence of the 31 species in the study site and its adjacent areas is confirmed by examination of the museum collection and recent field surveys (Table 5). Reads assigned to Lebbeus groenlandicus are considered to be cross-contamination apparently derived from previous experiments based on aquarium tank water (for details, see below).

A list of malacostracan species detected from natural sea water sampled at Banda, Tateyama, Chiba Prefecture. Non-decapod taxa are marked by bold. Number of reads, degree of sequence identity, length of marker sequences and accession numbers are summarised.

Family Species No. of reads Identity bp Acc. No.
Identity > 98%: 35 species
Portunidae Thranita pelsarti (Montgomery, 1931) 53,745 100 164 LC430787
Portunidae Thalamita sima H. Milne Edwards, 1834 32,026 100 164 LC430780
Xanthidae Paraxanthias elegans (Stimpson, 1858) 6.611 99.4 170 LC430814
Xanthidae Pilodius nigrocrinitus Stimpson, 1859 5,737 99.4 169 KM888688
Varunidae Gaetice depressus (De Haan, 1835) 5,732 100 169 LC469701
Alpheidae Betaeus granulimanus Yokoya, 1927 4,778 100 158 LC464541
Calappidae Calappa gallus (Herbst, 1803) 3,364 98.6 173 KU206589
Pilumnidae Pilumnus longicornis Hilgendorf, 1878 3,204 100 165 LC469661
Penaeidae Metapenaeopsis lamellata (Rathbun, 1902) 1,937 100 169 AF105043
Portunidae Thalamita seurati Nobili, 1906 1,757 98.2 169 LC469672
Xanthidae Leptodius affinis (Dana, 1852)* 1710 100 167 KP256206
Thoridae Heptacarpus futilirostris (Bate, 1888) 1,676 100 161 LC464531
Upogebiidae Tuerkayogebia kiiensis (Sakai, 1971) 1,391 100 174 LC472887
Majidae Micippa philyra (Herbst, 1803) 1,002 99.7 159 EU682780
Xanthidae Actaea semblatae Guinot, 1976 949 100 171 LC469679
Macrophthalmidae Chaenostoma orientale Stimpson, 1858** 914 100 168 AB101492
Xanthidae Cycloxanthops truncatus (De Haan, 1837) 779 100 169 LC430809
Majidae Tiarinia cornigera (Latreille, 1825) 635 99.7 162 EU682786
Grapsidae Pachygrapsus crassipes (Randall, 1840) 490 100 173 KC878511
Plagusiidae Guinusia dentipes (De Haan, 1835)*** 419 100 169 AJ308421
Paguridae Pagurus filholi (de Man, 1887) 310 100 168 LC464566
Epialtidae Pugettia quadridens (De Haan, 1839) 292 99.1 170 LC430812
Alpheidae Alpheus pacificus Dana, 1852 268 99.4 160 LC464538
Alpheidae Synalpheus tumidomanus (Paulson, 1875) 172 100 158 LC464544
Caprellidae Caprella scaura Templeton, 1836 89 99.3 134 AB847634
Eriphiidae Eriphia ferox Koh & Ng, 2008 79 100 150 HM637968
Diogenidae Aniculus miyakei Forest, 1984 70 100 167 LC430788
Sergestidae Sergia lucens (Hansen, 1919) 43 100 172 AB705159
Diogendiae Clibanarius virescens (Krauss, 1843) 39 98.8 170 LC464558
Xanthidae Microcassiope orientalis Takeda & Miyake, 1969 26 98.2 170 LC469689
Xanthidae Atergatis floridus (Linnaeus, 1767) 20 100 170 LC430782
Paguridae Pagurus maculosus Komai & Imafuku, 1996 16 100 168 LC464569
Grapsidae Pachygrapsus minutus A. Milne-Edwards, 1873 11 100 171 LC430755
Thoridae Lebbeus groenlandicus (Fabricius, 1775) 5 99.4 162 KJ833727
Xanthidae Gaillardiellus orientalis (Odhner, 1925) 4 100 169 LC469685
80% < Identity < 98%: 10 species
Majidae Micippa thalia (Herbst, 1803) 3,743 96.9 159 EU682780
Portunidae Thalamita sp. aff. coeruleipes 3,677 97 164 LC469674
Alpheidae Alpheus sp. aff. pacificus 1,321 81.3 160 LC472887
Pilumnidae Typhlocarcinus sp. 938 89.4 161 LC469664
Pilumnidae Pilumnus sp. 465 93.9 165 LC469663
Xanthidae Actaea polyacantha (Heller, 1861) 378 96.4 167 HM798416
Varunidae Sestrostoma depressum (Sakai, 1965) 225 87.4 175 LC469704
Pilumnidae Cryptolutea sagamiensis (Sakai, 1935) 189 89.4 161 LC430738
Maeridae Quadrimaera pacifica (Schellenberg, 1938) 179 84.1 126 AB432980
Euphausiidae Euphausia similis G.O. Sars, 1885 174 88.8 169 AF281274

A list of 90 decapod species recorded from rocky intertidal to shallow subtidal zones (< 5 m) in Tateyama Bay (including Banda) and nearby areas.

Infraorder Family Species CBM-ZC voucher 16S sequence eDNA detection
Caridea Alpheidae Alpheus bisincisus De Haan, 1849 14164 ND
Alpheus edwardsii (Audouin, 1826) 4674 ND
Alpheus heeia Banner & Banner, 1975 2965, 7010, 14816, 14820, 15088
Alpheus pacificus Dana, 1852 14830
Arete dorsalis (Stimpson, 1860) 371, 8210,
Athanas japonicus Kubo, 1936 2724, 5835, 14999
Athanas dimorphus Ortmann, 1894 5836, 7989, 14163 ND
Athanas parvus de Man, 1910 3641, 14818 ND
Betaeus gelasinifer Nomura & Komai, 2000 5209 ND
Betaeus granulimanus Yokoya, 1927 4673, 9481, 15239
Salmoneus gracilipes Miya, 1972 14185 ND
Synalpheus tumidomanus Paulson, 1875 11722, 15089
Crangonidae Syncrangon angusticauda (De Haan, 1849) 4937, 9937, 15001
Hippolytidae Hippolyte ventricosa H. Milne Edwards, 1837 6023, 9940 ND
Palaemonidae Cuapetes grandis (Stimpson, 1860) 14994
Palaemon pacificus (Stimpson, 1860) 15087, 15245
Palaemon serrifer (Stimpson, 1860) 540
Eualus sinensis (Yu, 1931) 9479 ND
Thoridae Heptacarpus futilirostris (Bate, 1888) 4672, 9480, 14162, 15148
Achelata Palinuridae Panulirus japonicus (von Siebold, 1824) 14991
Axiidea Axiidae Litoraxius boshu Komai & Tachikawa, 2007 9228
Callianassidae Nihonotrypaea petalura (Stimpson, 1860) 7011, 7990, 15003
Gebiidea Upogebiidae Tuerkayogebia kiiensis (Sakai, 1971) 9057, 15218
Anomura Galatheidae Galathea orientalis Stimpson, 1858 4679, 9500, 15014
Pachycheles hertwigi Balss, 1913 15015, 15091
Porcellanidae Pachycheles stevensii Stimpson, 1858 4678, 5143, 7828, 15016
Petrolisthes coccineus Owen, 1839 11289 ND
Petrolisthes japonicus (De Haan, 1849) 7580, 9938, 15017
Diogenidae Aniculus miyakei Forest, 1984 2584
Calcinus laevimanus (Randall, 1840) 690–692, 689
Calcinus latens (Randall, 1840) 698–703
Calcinus vachoni Forest, 1954 707–709, 15009
Clibanarius virescens (Krauss, 1843) 5141, 15008
Dardanus sanguinolentus (Quoy & Gaimard, 1824) 15233 ND
Paguristes ortmanni Miyake, 1978 15234
Paguridae Pagurus decimbranchiae Komai & Osawa, 2001 5897 ND
Pagurus erythrogrammus Komai, 2003 6496 ND
Pagurus filholi (de Man, 1887) 15010, 15161
Pagurus japonicus (Stimpson, 1858) 15011
Pagurus lanuginosus (De Haan, 1849) 4676 ND
Pagurus maculosus Komai & Imafuku, 1996 4677, 5142, 15012
Pagurus nigrivittatus Komai, 2003 6482–6484, 15013
Brachyura Calappidae Calappa gallus (Herbst, 1803) 15235
Inachidae Paratymolus pubescens Miers, 1879 7991 ND
Epialtidae Huenia heraldica (De Haan, 1837) 7992, 15094
Menaethius monoceros (Latreille, 1825) 15093
Pugettia quadridens (De Haan, 1849) 7581, 14821, 15018
Hymenosomatidae Elamena truncata (Stimpson, 1858) 11773 ND
Lucasinus coralicola (Rathbun, 1909) 3632 ND
Neohymenicus orientalis (Sakai, 1932) 4680 ND
Rhynchoplax messor Stimpson, 1858 4681, 6024, 9939, 11774 ND
Majidae Micippa philyra (Herbst, 1803) 2044, 15019
Brachyura Majidae Micippa thalia (Herbst, 1803) 11031
Pseudomicippe okamotoi (Sakai, 1938) 8017 ND
Tiarinia cornigera (Latreille, 1825) 7900, 15020
Eriphiidae Eriphia ferox Koh & Ng, 2008 2967, 15164
Portunidae Thalamita sima H. Milne Edwards, 1834 895, 2653, 13448
Thalamita seurati Nobili, 1906 13447
Thlanita pelsarti (Montgomery, 1931) 7582
Pilumnidae Benthopanope pearsei (Rathbun, 1932) 4683, 5148, 15241
Heteropilumnus ciliatus (Stimpson, 1858) 11291
Pilumnus longicornis Hilgendorf, 1878 11133, 12814, 12819, 14130
Pilumnus minutus De Haan, 1835 7997
Xanthidae Actaea semblatae Guinot, 1976 898, 12147, 14558
Atergatis floridus (Linnaeus, 1767) 11549, 15242
Cycloxanthops truncatus (H. Milne Edwards, 1834) 2611, 5144, 7995, 14822, 15021
Danielea noelensis (Ward, 1942) 15236
Etisus laevimanus (Randall, 1840) 2662
Gaillardiellus orientalis (Odhner, 1925) 8363
Forestiana granulata (Krauss, 1843) 15155
Leptodius affinis (Dana, 1852) 15022
Medaeops granulosus (Haswell, 1882) 7993, 11290, 13757, 14819
Microcassiope orientalis Takeda & Miyake, 1969 5145, 5838, 5987
Novactaea pulchella (A. Milne-Edwards, 1865) 5146 ND
Palapedia integra (De Haan, 1835) 7996 ND
Palapedia truncatifrons (Sakai, 1972) 13758, 15237
Paraxanthias elegans (Stimpson, 1858) 15023
Pilodius nigrocrinitus Stimpson, 1859 4682, 5147, 15096
Zozymodes cavipes (Dana, 1852) 15156 ND
Grapsidae Pachygrapsus crassipes (Randall, 1840) 7541, 15243
Pachygrapsus minutus A. Milne-Edwards, 1873 15157, 15165
Macrophthalmidae Chaenostoma orientale Stimpson, 1858 15167
Plagusiidae Guinusia dentipes (De Haan, 1835) 469, 564, 899, 7540
Sesarmidae Chiromantes haematocheir (De Haan, 1833) 2725
Nanosesarma minutum (de Man, 1887) 4685, 5149, 15166
Varunidae Gaetice depressus (De Haan, 1835) 15024, 15097
Hemigrapsus sanguineus (De Haan, 1835) 6762
Sestrostoma depressum (Sakai, 1965) 7012, 10545
Pinnotheridae Sakaina asiatica (Sakai, 1933) 6917 ND
Sakaina incisa Sakai, 1969 4684 ND

Sequences of the 69 no-hit taxa were subjected to BLAST search on GenBank database. None of them was assigned to crustacean species. Of the 69 taxa, five were assigned to the following three known molluscan species with high sequence identity: Patelloida saccharina (Lesson 1831) (Gastropoda: Lottiidae; one haplotype, sequence identity 100%); Limnoperna fortunei (Dunker 1857) (Bivalvia: Mytilidae; three haplotypes, sequence identity 99–100%); Mytilus galloprovincialis Lamarck, 1819 (Bivalvia: Mytilidae; one haplotype, sequence identity 100%). One taxon was assigned to a bryozoan Beania klugei Cook, 1968 (Gymnolaemata: Cheilostomatida: Beaniidae) with low sequence identity (93%). One taxon was linked to two unidentified cyanobacteria taxa with low sequence identity, i.e. Synechococcus sp. WH 8109 (sequence identity 95.5%) or Synechococcus sp. CC9605 (sequence identity 94.2%).

Discussion

Usefulness of eDNA metabarcoding with MiDeca primers

It has been confirmed that the newly developed MiDeca primers are able to amplify the hyper-variable region of the mitochondrial 16S rRNA gene from the tissue-derived DNA extracts. We have successfully sequenced the target segment from 250 species from 186 genera and 65 families distributed across 10 suborders/infraorders. The edit distance between species was very high (Fig. 3), suggesting that the MiDeca sequence has sufficient interspecific variations for taxonomic assignment. A preliminary examination of eDNA from the natural seawater from Banda, Tateyama, Chiba Prefecture, detected 34 decapod species (sequence identity > 98%) (Table 4). In addition to those species, 10 unidentified species with lower identity (80–98%) were also detected (Table 4). There is little doubt that the eDNA metabarcoding with the MiDeca primers could provide information on the presence of particular decapod species without the requirement for capturing specimens or visual census.

Taxa detected from eDNA metabarcoding

It is remarkable that as many as 34 decapod species were detected from only one sample. Of the 34 species detected, the occurrence of 32 species in the study site and nearby areas was confirmed by examination of the museum collections and our field surveys (Tables 4 and 5). Although voucher specimens have not been collected, the occurrence of Metapenaeopsis lamellata is still likely. This penaeid species is sublittoral and nocturnal and, thus, collection of specimens at the study site is difficult. The detection of the sergestid shrimp Sergia lucens is remarkable, because it is a mesopelagic species, undergoing diurnal vertical migration along the continental shelf (Omori 1969). This species sometimes occurs in coastal areas in Boso Peninsula (TK, personal observation; voucher material CBM-ZC 2053) and, thus, its detection was due either to an accidental migration to the coastal water or to transport of the eDNA from the nearby oceanic water.

The detection of the macrophthalmid crab “Macrophthalmus boscii” needs explanation. Recent studies have shown that more than one species were confounded under the name M. boscii (cf. Naderloo and Türkay 2011, Teng et al. 2016) and M. boscii and allied taxa were transferred to the genus Chaenostoma. Teng et al. (2016) showed that two species, C. crassimanus Stimpson, 1858 and C. orientale occur in the north-western Pacific, including Japan, with true C. boscii restricted to the western Indian Ocean. At the studied site, specimens of C. orientale have been collected and, thus, it is reasonable to consider that it was the eDNA of C. orientale that was detected and that the GenBank sequence identified as “Macrophthalmusboscii has been misidentified.

On the other hand, the detection of the thorid shrimp Lebbeus groenlandicus is dubious, because it is a boreal, deep-sea benthic species that does not occur in or adjacent to the study area. As such, we concurrently examined eDNA from tank water in Aquamarine Fukushima, where individuals of this species had been kept [for the species identity, see Komai (2015)]. This is, thus, the suspected source of “contamination”. This again highlights the importance of safeguarding against cross-contamination during the eDNA metabarcoding (e.g. Bohmann et al. 2014).

Amongst the eight decapod taxa with a lower identity match (80–98%), e-DNA assigned to the majid crab Micippa thalia (identity 96.9%) was also found. It is now known that the Japanese population that has been referred to as M. thalia in literature is actually a separate species (P. K. L., personal communication). As such, it is not surprising that the detected sequence here does not fully match that of M. thalia registered in GenBank. Although we have tried to sequence specimens identified as “M. thalia” in the CBM collection, we have not been successful. Unfortunately, our attempts to sequence specimens identified with M. thalia deposited in the CBM collection were unsuccessful.

The species status of the other seven decapod taxa with lower identity (80–98%) cannot be determined with a high degree of confidence. As shown in Table 5, there are several local species for which MiDeca sequence data are still not available. On the other hand, it is highly likely that there are species for which their occurrence has not been confirmed in the study area. At the study site, the intertidal to subtidal area consists of fragile sandstone, which provides cryptic habitats for those decapod species. To reduce the number of unknown species, continuous efforts for the collection and accumulation of reference sequences are necessary.

It is fortuitous that eDNA of the other malacostracan taxa, including Amphipoda (a caprellid Caprella scaura and an unidentified taxon linked to the maerid Quadrimaera pacifica) and Euphausiacea (an unidentified taxon linked to euphausiid Euphausia similis), were also detected (Table 4). In addition to those, taxa other than Crustacea were also detected, as noted above. This suggests broad applicability of MiDeca metabarcoding to non-decapod Malacostraca and even to non-crustacean taxa with slight modifications of primer sequences.

A false negative (species is not detected where it is present), as well as a false positive (species is detected where it is absent), are important issues in eDNA metabarcoding, because they will cause under- or overestimation of species richness. In fact, considerable numbers of decapod species that are recorded from the study site and adjacent areas were not detected by the present metabarcoding exercise (Table 5). Two major factors could be considered for the false negatives: 1) no eDNA from those species was collected during sampling; 2) PCR amplification of eDNA from those species was not successful.

With regard to the first factor, water sampling at low tide, including various microhabitats, may be more effective for collecting more eDNA. With regard to the second factor, exploration of an optimal method that generates a greater species richness of MiDeca sequences to avoid PCR dropouts would be necessary. As PCR dropouts might be due to PCR bias derived from primer-template mismatches, an optimal number of PCR replicates and use of multiple annealing temperatures would be alternative approaches to comprehensively detect target eDNA. In fact, in a fungal metabarcoding study, pooling multiple repeated PCRs and using multiple annealing temperatures were recommended to facilitate the recovery of more accurate species richness (Schmidt et al. 2013). Furthermore, Majaneva et al. (2018) demonstrated that choice of the DNA extraction method affects DNA metabarcoding. Clarification of the optimal DNA extraction method for the target group might be advisable.

The other important point for accurate assessment of biodiversity is completeness of the reference sequence database, which is indispensable for satisfactory taxonomic assignments. Reference sequences, used in the present analyses, were primarily derived from the GenBank database with the addition of the sequence data generated by ourselves. The process of exploration of the GenBank decapod crustacean database highlighted the lack of sufficient sequence data. During this study, we have newly sequenced the target marker from 250 decapod species (including 4 unidentified species), but the number of the currently available species known from Japanese waters is still 1054, representing 36% of the decapod species presently known from the areas (about 2,890 species placed in 950 genera distributed across 147 families). Furthermore, there are many species with uncertain taxonomy, including undescribed species and poorly defined species, for example, taxa in the snapping shrimp genus Alpheus (cf. Anker and De Grave 2016), swimming crabs of the genus Thalamita (cf. Spiridonov 2017) and several genera in the highly diverse crab family Pilumnidae (cf. Ng 1987). As with standard DNA barcoding, significantly more taxonomic work, including building of verified reference sequence databases, is necessary to optimise the effectiveness of eDNA approaches.

Data accessibility

MiDeca sequences from the 250 decapod crustaceans are available from DDBJ/EMBL/GenBank databases (Table 2). Raw reads from the MiSeq sequencing are available from the DDBJ Sequence Read Archive (DRA008193/DRR172676).

Acknowledgements

TK was supported by JSPS KAKENHI Grant Number 16K07494 and MM was supported by JST CREST Grant Number JPMJCR13A2, Japan and by Environment Research and Technology Development Fund (4-1602) of the Ministry of the Environment, Japan. We deeply thank Shoji Tsuzuki and Ryuta Yoshida (Tateyama Station, Ochanomizu University) for providing us with valuable specimens for this study, Takehiko Fukuchi for assisting in obtaining reference sequences of the 16S rRNA and two referees for reviewing the manuscript and for offering valuable comments for improvements. Shane T. Ahyong (Australian Museum, Sydney), Peter K. L. Ng (Lee Kong Chian Natural History Museum, National University of Singapore) and Ling-Ming Tsang (National Taiwan Ocean University, Keelung) also kindly reviewed and edited the manuscript.

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