Molecular identification and larval morphology of spionid polychaetes (Annelida, Spionidae) from northeastern Japan

Abstract Planktonic larvae of spionid polychaetes are among the most common and abundant group in coastal meroplankton worldwide. The present study reports the morphology of spionid larvae collected mainly from coastal waters of northeastern Japan that were identified by the comparison of adult and larval 18S and 16S rRNA gene sequences. The molecular analysis effectively discriminated the species. Adult sequences of 48 species from 14 genera (Aonides Claparède, 1864; Boccardia Carazzi, 1893; Boccardiella Blake & Kudenov, 1978; Dipolydora Verrill, 1881; Laonice Malmgren, 1867; Malacoceros Quatrefages, 1843; Paraprionospio Caullery, 1914; Polydora Bosc, 1802; Prionospio Malmgren, 1867; Pseudopolydora Czerniavsky, 1881; Rhynchospio Hartman, 1936; Scolelepis Blainville, 1828; Spio Fabricius, 1785; Spiophanes Grube, 1860) and larval sequences of 41 species from 14 genera (Aonides; Boccardia; Boccardiella; Dipolydora; Laonice; Paraprionospio; Poecilochaetus Claparède in Ehlers, 1875; Polydora; Prionospio; Pseudopolydora; Rhynchospio; Scolelepis; Spio; Spiophanes) of spionid polychaetes were obtained; sequences of 27 of these species matched between adults and larvae. Morphology of the larvae was generally species‐specific, and larvae from the same genus mostly shared morphological features, with some exceptions. Color and number of eyes, overall body shape, and type and arrangement of pigmentation are the most obvious differences between genera or species. The morphological information on spionid larvae provided in this study contributes to species or genus level larval identification of this taxon in the studied area. Identification keys to genera and species of planktonic spionid larvae in northeastern Japan are provided. The preliminary results of the molecular phylogeny of the family Spionidae using 18S and 16S rRNA gene regions are also provided.

Many marine invertebrates including polychaetes pass through a planktonic larval phase during their early life history. As such, planktonic larvae derived from the benthic polychaetes are one of the most numerous and diverse groups of coastal zooplankton (Thorson 1946;Hansen 1999;Omel'yanenko and Kulikova 2002;Blake 2017). Polychaetes are often represented in coastal benthic fauna with high species richness all over the world, and members of this group play a large role in the functioning and food webs of marine ecosystems (Aller 1982;Laffaille et al. 2005;Tomiyama et al. 2005Tomiyama et al. , 2007. However, the field study of larval ecology has been restricted because of difficulties in larval identification, which is largely caused by the radical morphological differences between larval and adult stages, lack of diagnostic key characters of larvae, and lack of information on larval forms of many species as is also the case for other marine invertebrates (Branscomb and Vedder 1982;Shanks 1986;Levin 1990). Extensive efforts to describe planktonic polychaete larvae from coastal waters have been performed for species from European and American waters (Blake 2017, and the references cited therein). However, most of these studies are very limited regarding other areas, although Carrasco (1976) and Wu et al. (1978) described polychaete larvae of many species from Chilean and Chinese waters, respectively. In Japan, the larval development and morphology of some polychaete species have been studied (e.g., Izuka 1912: Nereididae Blainville, 1818Okada 1930: Syllidae Grube, 1850Okuda 1946: 9 families;Imajima 1959: Spionidae Grube, 1850Choe 1960: Eunicidae Berthold, 1827Yamaji 1966: 16 families;Tokioka 1970: Amphinomidae Lamarck, 1818Imai 1975Miura andKajihara 1981: Serpulidae Rafinesque, 1815;Yokoyama 1981Sasaki andBrown 1983: Saccocirridae Bobretzky, 1872;Yokouchi 1985Yokouchi , 1988Sato and Tsuchiya 1991: Nereididae;Nishi and Yamasu 1992a, 1992b, 1992c, 1992dYokouchi and Yokouchi 1997: 23 families; Koya et al. 2003: Nereididae;Tosuji and Sato 2006: Nereididae;Kondoh et al. 2017: Spionidae;Kan et al. 2020: Nereididae), and some field ecological investigations of polychaete larvae have been conducted (Yokouchi 1984(Yokouchi , 1991Yokoyama 1990Yokoyama , 1995Abe et al. 2011Abe et al. , 2014Kan et al. 2020).
Spionidae is one of the largest taxa of polychaete annelids and currently comprises more than 500 nominal species belonging to approximately 38 genera (Radashevsky 2012;Read and Fauchald 2020, excluding Poecilochaetus Claparède in Ehlers 1875and Trochochaeta Levinsen, 1883. Planktonic spionid larvae are often the most common and abundant group in the coastal meroplankton (Anger et al. 1986;Levin 1986;Abe et al. 2011Abe et al. , 2014 because of their high abundance and species richness in coastal zones, high reproductive capacity, and relatively long planktonic stage (Blake 1996;Blake and Arnofsky 1999). As they are often seasonally dominant in coastal zooplankton communities, spionid larvae can play a major role in planktonic trophic dynamics (Martin et al. 1996;Pedersen et al. 2010). They are also reported to constitute a large portion of ballast water species (Carlton and Geller 1993;Carlton 1996). Spionidae includes species that adult inhabit a wide range of substrates and some are symbionts of other invertebrates Britayev 1998, 2018;Sato-Okoshi 1999Abe et al. 2019b). Among these, symbionts polydorids (i.e., from the Polydora complex or tribe Polydorini, see Radashevsky 2012) are well known as harmful pests in molluscan aquaculture because of their shell boring activities (Blake and Evans 1972;Handley and Bergquist 1997;Simon et al. 2006;Simon and Sato-Okoshi 2015). Understanding larval dynamics and dispersal is important to prevent the settlement of pest spionid species on the shells of aquaculture mollusks (Sato-Okoshi et al. 1990;Simon 2015;David et al. 2016). The host/substrate selectivity and settlement mechanism of spionid larvae during their developmental process are also interesting aspects of larval biology. Although various morphological characteristics of larvae including body shape, pigment patterns (placement and number), ciliary organization, and to some extent, chaetae can be generally used to identify the planktonic larvae of spionid species (Blake and Arnofsky 1999), species-level identification is still difficult because of the lack of information on the larval forms of many species.
The link between larval and adult form has been traditionally achieved by labor-intensive culturing approaches either through rearing larvae collected from plankton or by spawning adults in the laboratory (Shanks 2001). In recent years, ecological studies on the diversity and distribution of marine planktonic larvae are increasingly depending on molecular methods for accurate taxonomic identification to species level (Andre et al. 1999;Hosoi et al. 2004;Pradillon et al. 2007;Phillips et al. 2008;Heimeier et al. 2010). For future metabarcoding studies, establishment of a comprehensive DNA barcoding library is very useful for rapid identification of planktonic larvae. Meanwhile, the use of molecular methods for identifying planktonic larvae in extensive field surveys handling large numbers of collected samples still requires extensive cost. Since the direct microscopic observation, which allows prompt identification of larvae at low cost, remains the popular technique for distinguishing planktonic larvae, information on larval morphology would be useful for such studies.
The aim of the present study is identification of spionid larvae that dominantly appear among the planktonic polychaete larvae from northeastern Japan (Abe et al. 2011(Abe et al. , 2014 by comparing adult and larval gene sequences. The 18S rRNA and 16S rRNA genes was herein used as a marker for species-level discrimination in Spionidae. Moreover, we report the results of preliminary phylogenetic analysis using these genetic regions and describe the morphologies of spionid larvae with photomicrographs of living specimens.

DNA analysis and larval identification
Adults and larvae of one or more individuals, respectively, were subjected to DNA analysis. In order to clarify the development links between the different stages, we analyzed the DNA of as many larvae of different stages as possible. Except for Laonice sp. 2 (Fig. 4E), all larvae pictured in this paper have been identified by DNA analysis. All individuals were washed by several transfers in sterile filtered (pore size 0.2 µm) seawater and distilled water to remove as much extraneous matter as possible before DNA extraction. Genomic DNA was extracted from live or ethanol-preserved larval (from the whole body) and adult (from palp or a small piece of tissue) spionid specimens by grinding and heating at 95 °C for 20 min in 50 µl TE buffer (pH 8.0) with 10% Chelex 100 (Bio-Rad; Richlen and Barber 2005). Undiluted or 10-fold diluted extracted DNA in TE buffer was used as template for polymerase chain reaction (PCR) depending on the DNA concentration. Partial sequences of nuclear 18S rRNA gene were amplified by PCR according to the methods described by Abe (2012, 2013) and Teramoto et al. (2013) using the following primer pairs (Nishitani et al. 2012): 18S-1F1 (AACCTGGTTKATCCTGCCAG) and 18S-1R632 (ACTAC-GAGCTTTTTAACYGCARC), 18S-2F576 (GGTAATTCCAGCTCYAATRG) and 18S-2R1209 (AAGTTTYCCCGTGTTGARTC), and 18S-3F1129 (GCTGAAACT-TAAAGRAATTGACGG) and 18S-R1772 (TCACCTACGGAAACCTTGTTACG). Partial sequences of mitochondrial 16S rRNA gene were amplified by PCR according to the methods described by Abe et al. (2019a) using the 16Sar (CGCCTGTT-TATCAAAAACAT) and 16Sbr (CCGGTCTGAACTCAGATCACGT) primer pair (Palumbi et al. 1991). The PCR products were purified using ExoSAP-IT (Affymetrix, Cleveland, OH, USA) and sequenced by Eurofins Genomics (Tokyo, Japan). The forward and reverse complementary sequences and contigs were assembled using GeneStudio ver. 2.2.0.0 (GeneStudio, Inc. Suwanee, GA, USA). Larval and adult gene sequences obtained in the present study (Table 1) were aligned using the MAFFT online service ver. 7 with the L-INS-i algorithm (Katoh et al. 2017) with (Fig. 2) and without (Fig. 3) the sequences of other spionid species available in the DNA Data Bank of Japan (DDBJ), the European Nucleotide Archive (ENA), or GenBank databases (  Rousset et al. (2007) aLRT) with 5,000 replicates (Guindon et al. 2010), approximate Bayes (aBayes) test (Anisimova et al. 2011), and ultrafast bootstraps (UFBoot) with 5000 replicates (Hoang et al. 2018). SH-aLRT ≥ 80%, aBayes ≥ 0.95, and UFBoot ≥ 95% were defined as robust statistical support. All the sequences newly generated in this study were deposited in the DDBJ/ENA/GenBank nucleotide sequence database under accession numbers LC545853 to LC545925 and LC595683 to LC595767 (Table 1). Part of the sequences used in the present study was reported in the previous studies (see Table 1). The planktonic spionid larvae were identified by comparing larval and adult sequences obtained in the present study and/or by larval morphology.

results
The 18S and 16S rRNA gene analyses of larval and adult spionids Nuclear 18S and mitochondrial 16S rRNA gene sequences of adult spionid polychaetes were successfully obtained from 48 species belonging to 14 genera (Table 1).
In the phylogenetic analysis using only the sequences obtained in the present study (i.e., without DBJ/ENA/GenBank sequences), species from the genera Paraprionospio, Pseudopolydora, Scolelepis, Spio, and Spiophanes were recovered as monophyletic groups with robust statistical supports (i.e., SH-aLRT ≥ 80%, aBayes ≥ 0.95, and UFBoot ≥ 95%, Fig. 2). Species belonging to the genus Boccardia except for B. pseudonatrix and those from the genus Dipolydora except for D. armata and D. cf. commensalis were recovered as monophyletic groups with robust statistical supports. The tribe Polydorini and subfamily Spioninae were also recovered as monophyletic although UFBoot of monophyly of the subfamily Spioninae was with low support (≤ 95%).
In the phylogenetic analysis with the sequences obtained in the present study and from DDBJ/ENA/GenBank databases, species belonging to the genera Poecilochaetus, Laonice, Marenzelleria, Pseudopolydora, Pygospio, Rhynchospio, Scolelepis, Spio + Microspio, and Spiophanes, were recovered as monophyletic groups with robust statistical supports (Fig. 3). Species belonging to the genus Polydora were recovered as a monophyletic group but with low UFBoot support. Tribe Polydorini + Pygospio, that plus Glandulospio, and Spiophanes + Trochochaeta were also recovered as monophyletic groups with robust statistical supports. The genera Poecilochaetus and Trochochaeta, which were previously considered as belonging to the family Poecilochaetidae and Trochochaetidae, respectively, were recovered as ingroup taxa of the family Spionidae with robust statistical supports (Fig. 3).
In total, 41 species belonging to 14 genera of planktonic spionid larvae were identified (Table 1; Fig. 2), 27 of which were identified by the 100% or nearly 100% match between the sequences obtained from adult and larvae (Fig. 2). The other 14 species of spionid larvae were identified to species or genus level based on their phylogenetic position and/or larval morphology. Tentative larval diagnosis for each genus and larval identification keys to species of each genus based on the morphol-  Table 1). The gene sequences of adult and larval spionid polychaetes are indicated by solid squares and circles in front of each species name, respectively. SH-aLRT/approximate Bayes support/ultrafast bootstrap support values of ≥ 80%, ≥ 0.95, ≥ 95%, respectively are given beside the respective nodes. Nodes with red circles indicate triple high support values of SH-aLRT ≥ 80, approximate Bayes support ≥ 0.95, and ultrafast bootstrap support ≥ 95. The scale bar represents the number of substitutions per site. Sequences of Laonome sp. and Sabella pavonina Savigny, 1822 obtained from DDBJ/EMBL/GenBank were used for outgroup rooting.  Table 1) and from DDBJ/EMBL/GenBank (shown in Table 2). The tree is divided into two parts A, B. The gene sequences obtained in the present study are highlighted by bold and red color and the adult and larval sequences are indicated by solid squares and circles in front of each species name, respectively. SH-aLRT/approximate Bayes support/ ultrafast bootstrap support values of ≥80%/≥0.95/≥95%, respectively are given beside the respective nodes. Nodes with red circles indicate triple high support values of SH-aLRT ≥ 80, approximate Bayes support ≥ 0.95, and ultrafast bootstrap support ≥ 95. The scale bar represents the number of substitutions per site. Sequences of Laonome sp. and Sabella pavonina Savigny, 1822 obtained from GenBank were used for outgroup rooting.
ogy of late spionid larvae from northeastern Japan are provided at the beginning of the larval morphological description section of each genus. An identification key to genera is provided below. Identification key to genera of the spionid larvae in northeastern Japan Larval diagnosis. The overall shape slender. Prostomium rounded or rectangular anteriorly. The lateral parts of the peristomium more or less demarcated from prostomium. Two pairs of red eyes present. Melanophore absent, some brown or dark pigmentation may be present in pharynx and pygidium. Larval chaetae coarsely or slightly serrated. Larval chaetae in first chaetiger very long, extend beyond pygidium in late trochophore and early nectochaete stages. Nototrochs develop in late larval stages. Gastrotrochs occur in all chaetigers from chaetiger II onwards. Two pairs of pygidial cirri develop in late larval stage. The body of early larvae covered by egg envelope, yellowish opaque appearance with abundant yolk. Two parallel rows of encircling vesicles of egg envelope present in pretrochophore and trochophore stages. Holopelagic lecithotrophic development unique among spionids (Hannerz 1956;Arnofsky 1999, as Dispio uncinata Hartman, 1951: see Radashevsky et al. 2011;Blake 2006, as D. uncinata). In ten-chaetiger larvae, egg envelope becomes incorporated into larval cuticle, two pairs of red eyes arranged in an approximately straight line (Fig. 4B). Larval chaetae on first chaetiger long especially in early larvae (Fig. 4A, B). Late larvae long and slender in shape (Fig. 4C). Prostomium rectangular anteriorly in larval stages, considerably ex-tended and tapered in juvenile stage. Lateral parts of peristomium moderately demarcated from prostomium. Black pigment in pharynx. Pigmentation absent except in the eyes and pharynx. Pygidium acquires two pairs of dorsal cirri in late larvae (Fig. 4C).
Remarks. Adult individuals of this species were collected from muddy bottom sediment at 22 m depth in Onagawa Bay in January 2011 and 2012 using a Smith-McIntyre grab sampler. Adult morphology agrees with the description of A. oxycephala by Imajima (1989). Aonides oxycephala originally described from Norway has been reported worldwide and is considered cosmopolitan. However, these reports may comprise a series of similar or sibling species, as pointed out by Radashevsky (2015). The gene sequences obtained in the present study were 100% match in 18S rRNA but 8.6% (29/337 bp) different in 16S rRNA from that of A. oxycephala from France (MG913226 and MG878895). Therefore, the species collected in the present study was referred to A. aff. oxycephala. The larvae and adults were confirmed to match (18S: 1753/1753, 16S: 447/448 bp) using molecular data (Fig. 2).
Planktonic larvae were found in Onagawa Bay from October to December. In early larval stages, the larvae of this species are similar to those of Laonice sp. (Fig. 4D); but larval chaetae are longer, and the body is yolkier and opaquer in this species. The larval morphology of A. aff. oxycephala is similar to that of A. oxycephala described by Hannerz (1956). However, the peristomium of the former species is more developed and demarcated from the prostomium compared to the latter. Black pigmentation of the pharynx in late larval stages was not reported by Hannerz (1956).

Genus Laonice Malmgren, 1867
Larval diagnosis. Overall shape short, thick, and fusiform. Prostomium stumpy, rectangular, notched anteriorly. Lateral parts of peristomium clearly demarcated from prostomium. The short palps attached to outer end of lateral parts of peristomium. Two pairs of red eyes present. Melanophores and pigmentation absent except eyes. Nototrochs absent. Gastrotrochs occur in all chaetigers from chaetiger III onwards. Well-developed serrated larval chaetae occur both in noto-and neuropodia, notochaetae characteristically introverted toward medial line of dorsal side. Early larvae covered by egg envelope (Hannerz 1956;Plate and Husemann 1994).
Laonice sp. 1 Fig. 4D Larval morphology. Remnants of egg envelope apparent in early trochophore (Fig. 4D). Two pairs of red eyes located in approximately a straight line, lateralmost pair larger in early larvae. Parapodia weakly differentiated; serrated larval chaetae introverted toward medial line of dorsal side. The body opaque yellowish with abundant yolk internally. Pigmentation absent except in the eyes.

Remarks.
Adult individuals of this species were collected from bottom sediments at 22 m depth in Onagawa Bay in December 2011 using a Smith-McIntyre grab sampler. To date, two Laonice species, L. cirrata (Sars, 1851) and L. japonica (Moore, 1907) have been recorded from Japan. Sikorski (2002) indicated that L. cirrata, a previously presumed widespread species, is probably limited to Norway and adjacent regions. This was supported by a molecular study that suggested previously unrecognized diversity within this species (Bogantes et al. 2018). Laonice japonica, originally described as Spionides japonicus from Japan and later considered as synonymous with L. cirrata (e.g., Söderström 1920;Berkeley and Berkeley 1936;Okuda 1937;Imajima and Hartman 1964;Foster 1971), was reexamined and considered a valid species by Maciolek (2000) and Sikorski (2011). However, even after that, since L. cirrata has been recorded from Japan (e.g., Imajima 2006Imajima , 2009Imajima , 2011, the validity of these records is ambiguous and might represent different species. In addition to these two species, unidentified Laonice sp. was also reported from Japan by Imajima (1990c) but it is unclear whether the species is identical to the species reported here. Although the 18S rRNA gene sequences obtained in the present study match (1731/1731 bp, except for gaps) with Laonice cirrata sequences from Russia in DDBJ/EMBL/GenBank (KM998754), because taxonomic knowledge on this genus in Japan is still limited and the 18S rRNA gene is relatively conservative, this species was referred to Laonice sp. The larvae and adults were confirmed to match (18S: 1754/1754, 16S: 500/504 bp) using molecular data (Fig. 3).
Larvae of this species were rare in the planktonic community found in Onagawa Bay in September 2011 and October 2012. Although two parallel rows of encircling vesicles of egg envelope, similar to those of Aonides pretrochophore and trochophore stages, were reported in oocytes of Laonice species (Radashevsky and Lana 2009), this characteristic was not observed in early larval stages with egg envelope in the present study ( Fig. 4D). Blake (2006) described the larval development of Laonice sp. from California; however, the identification of these larvae is doubtful because they seem to lack serrated larval notochaetae introverted toward the medial line of the dorsal side, which are characteristic of Laonice larvae.

Laonice sp. 2 Fig. 4E
Larval morphology. Late larvae thick and stumpy in shape (Fig. 4E). Prostomium stumpy, somewhat notched at tip. Lateral parts of peristomium well demarcated from prostomium. In late larvae, two pairs of red eyes are arranged in a trapezoidal shape, the medial pair bigger and situated anteriorly. Short palps developed in late larvae, attached to outer end of lateral parts of peristomium. Parapodia weakly differentiated; serrated larval chaetae in both noto-and neuropodia; notochaetae characteristically introverted toward medial line of dorsal side. Gut dark green in color internally. Pigmentation absent except in the eyes. Remarks. Adult individuals of this species were not collected in the present study. Only one individual of larva of this species were collected in Habu Port in June 2016. Even though the 18S and 16S rRNA gene sequences were not obtained, the larvae were identified as belonging to Laonice because the larval morphology of this species agrees with that of L. cirrata described by Hannerz (1956) and L. cf. cirrata described by Plate and Husemann (1994). However, the hooded hooks in neuropodia described by Hannerz (1956) were not observed in the specimens of the present study (nor in those reported by Plate and Husemann [1994]); this may be because the larvae collected here were less developed and the hooks were reported only from chaetiger XIV onwards.

Genus Paraprionospio Caullery, 1914
Larval diagnosis. Overall shape long and slender, large in size (> 4 mm) and number of chaetigers (> 35 chaetigers) at metamorphosis. Prostomium rounded. Lateral parts of peristomium moderately demarcated from prostomium. Two pairs of red or dark red eyes present. Pigmentation absent except eyes and some reddish pigmentation on pygidium. Nototrochs absent. Gastrotrochs occur in all chaetigers from chaetiger II onwards. Branchiae well developed and elongated in late larvae. Long larval chaetae may be absent in chaetiger II (Berkeley and Berkeley 1961, as Prionospio;Carrasco 1976;Yokoyama 1981Yokoyama , 1996Blake and Arnofsky 1999). Wilson, 1990 Fig. 5A, B Larval morphology. Long and thin in shape, quite large and long body with numerous chaetigers. Prostomium anteriorly rounded, lateral lips elevated from the ventrolateral side of prostomium (Fig. 5A). Late larvae acquire caruncle extending posteriorly from posterior part of prostomium (Fig. 5B). Peristomium fuses with the first larval segment at late larval stage (Fig. 5B). First pair of branchiae well developed, branchial pinnation still absent. Two pairs of red eyes arranged in somewhat trapezoidal shape, lateral pair kidney-shaped, situated anteriorly. Posterior part of pygidium pigmented reddish brown, anal cirri develop in late larvae.

Paraprionospio coora
Remarks. Adult individuals of this species were collected from muddy bottom sediments at 22 m depth in Onagawa Bay in December 2011 by using a Smith-McIntyre grab sampler. Adult morphology agrees with the description of P. coora by Yokoyama (2007), and therefore this species was referred to P. coora. The larvae and adults were confirmed to match (18S: 1754/1754, 16S: 500/500 bp) using molecular data (Fig. 2).
Only three planktonic larvae of this species were found in Onagawa Bay in November 2011 and Sasuhama in January 2013. The morphological characteristics and size of these larvae are similar to those in previous descriptions of the species from the same genus (Yokoyama 1981(Yokoyama , 1996. However, the larvae of P. coora lack red pigmentation on the dorsolateral side of the lateral lips, which characterizes the larvae of Paraprionospio patiens Yokoyama, 2007(Yokoyama 1981as P. pinnata: see Yokoyama 2007). Additionally, lamellae of the first pair of branchiae in P. coora are less developed in late larvae with more than 30 chaetigers (Fig. 5B) compared with the larvae of P. patiens (Yokoyama 1981) and Paraprionospio cordifolia Yokoyama, 2007(Yokoyama 1996, as Paraprionospio sp. form B: see Yokoyama 2007). Yokoyama (1981) suggested that the larvae of Paraprionospio are the largest in size and number of chaetigers at metamorphosis among the spionid larvae. However, late larvae of Poecilochaetus exceeding 5 mm (Magalhães et al. 2015) and with more than 40 chaetigers are often reported (Hannerz 1956;Plate and Husemann 1994).

Genus Poecilochaetus Claparède in Ehlers, 1875
Larval diagnosis. Overall shape long and slender, large in size (> 5 mm) and number of chaetigers (> 30 chaetigers) at metamorphosis. Body transparent, characterized by total absence of pigmentation except pairs of small pigment spot between parapodia or ventro-lateral side of each chaetiger. Two pairs of red or dark red eyes present. Gastrotrochs from chaetiger I onwards in all following chaetigers, gastrotrochs in first and second chaetigers represented by solitary lateral patches of cilia and complete gastrotrochs occur from third chaetiger onwards. Nototrochs absent. Larvae prior to ca. 30-40 chaetiger stages remain in metatrochophore stage, characterized by absence of functional parapodia for swimming and presence of well-developed proto-, telo-, and gastrotrochs for swimming. Metatrochophore have broadened trapezoidal prostomium with tactile cilia in anterior part, broad and low caruncle, provisional larval chaetae, pygidium without anal cirri. Larval stage after metatrochophore stage (often called nectosoma) characterized by reduced trochs, the presence of functional parapodia, and rapid serpentine swimming behavior. In nectosoma stage, caruncle, nuchal lobes, a pair of palps, parapodia, cirriform or digitiform dorsal and ventral postchaetal lobes, and two pairs of anal cirri on pygidium develop gradually (Thorson 1946;Hannerz 1956;Berkeley and Berkeley 1961;Reddy and Mohan 1982;Plate and Husemann 1994;Magalhães et al. 2015).
Poecilochaetus sp. Fig.4F Larval morphology. Overall shape long and slender. Two pairs of dark red eyes present. Metatrochophore larvae with 17 chaetigers have broadened trapezoidal prostomium with tactile cilia in anterior part, broad and low caruncle, provisional larval chaetae, and well developed prototrochs (Fig. 4F). Body of metatrochophore larvae transparent, characterized by small pigment spot between parapodia from chaetiger II onwards. Pygidium without anal cirri. Incomplete anterior fragment of nectosoma larvae characterized by extremely long body, rounded prostomium, broad and low caruncle, reduced prototrochs, parapodia with digitiform dorsal postchaetal lobes, and rapid serpentine swimming behavior. The body of nectosoma larvae transparent and small pigment spot laterally on each side of chaetigers. Occipital antenna and pair of palps not observed.
Remarks. Adult individuals of this species were not collected in the present study. Even though the 18S and 16S rRNA gene sequences obtained from larvae in the present study did not match any of the Poecilochaetus sequences from DDBJ/EMBL/ GenBank, this species was referred to Poecilochaetus sp. because specimens formed a monophyletic clade with the other Poecilochaetus species with robust statistical support (Fig. 3).
Planktonic larvae of this species were collected in Onagawa Bay in August 2010 and January 2013. The larval morphology of this species is similar to that of Poecilochaetus serpens Allen, 1904 described by Hannerz (1956) and Plate and Husemann (1994). However, the former species differs from the latter by not having yellow chromatophores on the "head" and pygidium as described by Plate and Husemann (1994). The pigmentation pattern of Poecilochaetus sp. larvae also differs from that of Poecilochaetus anterospinus, which has a pair of ventral green melanophores on the lateral side of each segment, beginning from chaetiger IV throughout (Magalhães et al. 2015).

Genus Prionospio Malmgren, 1867
Larval diagnosis. Overall shape long and slender. Prostomium rounded anteriorly. Lateral parts of peristomium not demarcated from the prostomium. Two pairs of red or dark red eyes present. Pigmentation usually absent except for eyes and on pygidium. Some species (e.g., P. steenstrupi and P. krusadensis) have red or green pigmentation on pharynx, dorsal side, and/or pygidium. Nototrochs absent or occur in branchial chaetigers in late larvae. Gastrotrochs occur in all chaetigers from chaetiger II-IV onwards (Thorson 1946, as Disoma;Hannerz 1956;Plate and Husemann 1994;. Identification key to species of the larvae belonging to the genus Prionospio in northeastern Japan Larval morphology. Long and thin in shape. Prostomium rounded anteriorly, lateral parts of the peristomium not especially well demarcated. Two pairs of red eyes arranged somewhat in trapezoidal shape, lateral pair kidney-shaped and situated anteriorly. Body extremely transparent (Fig. 5D). Green pigmentation on pharynx and pygidium (Fig. 5C, D). Caruncle develop in late larvae, extends posteriorly from posterior part of prostomium (Fig. 5C). In late larvae, branchial anlages occur from chaetiger II, pygidium acquires anal cirri.
Remarks. Adult individuals of this species were collected from shallow subtidal muddy bottom sediments in Sasuhama in August 2011 by using a hand-scoop. The species was referred to P. krusadensis as the adult morphology agrees with the descriptions of this species by Imajima (1990a). The larvae and adults were confirmed to match (18S: 1749/1749, 16S: 506/507 bp) using molecular data (Fig. 2).
Planktonic larvae of this species were found in Sasuhama and Onagawa Bay in July and August, but they were rare in the plankton samples. Green pigmentation on the pharynx and pygidium is characteristic of the larvae of this species. Imajima, 1990 Fig. 5E-G Larval morphology. Long and thin in shape. Prostomium rounded anteriorly, lateral parts of peristomium not especially well demarcated. Two pairs of red or dark red eyes arranged somewhat in trapezoidal shape, lateral pair kidney-shaped and situated anteriorly. Small caruncle develop in late larvae, extends posteriorly from posterior part of prostomium (Fig. 5G). In late larvae branchial anlages occur from chaetiger II, pygidium acquires anal cirri (Fig. 5G).
Remarks. Adult individuals of this species were collected from muddy bottom sediments at 22 m depth in Onagawa Bay in December 2011 by using a Smith-McIntyre grab sampler. The species was referred to P. membranacea as the adult morphology agrees with the descriptions of this species by Imajima (1990b). The larvae and adults were confirmed to match (18S: 1752/1752 and 1747/1750 except for gaps, 16S: 502/505 bp) using molecular data (Fig. 2). Planktonic larvae of this species were found in Onagawa Bay during August to October.
Prionospio spp. 1 and 2 Fig. 5H, I Larval morphology. Long and thin in shape. Prostomium rounded anteriorly, lateral parts of the peristomium not especially well demarcated. Two pairs of red or dark red eyes arranged somewhat in trapezoidal shape, lateral pair kidney-shaped and situated anteriorly. Small caruncle develop in late larvae, extends posteriorly from posterior part of the prostomium. In Prionospio sp. 2, branchial anlages occur from chaetiger II, pygidium acquires anal cirri (Fig. 5I). Pigmentation absent except eyes in Prionospio sp. 1. Gut pigmented in orange in Prionospio sp. 2. Palps developed in 19-chaetiger larva of Prionospio sp. 2.
Remarks. Two unidentified species of planktonic larvae of the genus Prionospio other than P. krusadensis and P. membranacea were collected from Onagawa Bay. The adult individuals of these species were not collected in the present study. Even though the 18S and 16S rRNA gene sequences obtained from these larvae in the present study did not match any Prionospio sequences obtained in the present study nor with those registered in DDBJ/EMBL/GenBank, these species were referred to Prionospio sp. 1 and 2 as the larvae were similar to the other Prionospio species in their morphology and gene sequences (Figs 2, 3). The larvae of Prionospio sp. 1 and 2 are similar to each other and to that of P. membranacea, and it is difficult to distinguish among them based only on their morphology.

Genus Rhynchospio Hartman, 1936
Larval diagnosis. Overall body shape short and thick. Prostomium broad and straight or slightly notched anteriorly. Lateral parts of peristomium clearly demarcated from prostomium. Palps attached to outer end of lateral parts of peristomium. Two pairs of red or black eyes present. Faint yellow pigment may be present in anterior part of prostomium and posterior part of pygidium. In late larvae, pair of prominent anterolateral processes on prostomium developed. Melanophore absent, black or yellowish pigmentation occur in some species. Nototrochs weakly developed, occur in all chaetigers except first chaetiger. Gastrotrochs occur regularly in every other chaetiger from chaetiger III onwards (Carrasco 1976;Radashevsky 2007 Larval morphology. Overall body shape short and thick in relation to length. Prostomium broad, stumpy, somewhat notched anteriorly. Peristomium well developed, forming wide collar on sides of prostomium. Two pairs of red or dark red eyes arranged in straight line, lateral pair in kidney-shape. Parapodia strongly differentiated in late larvae. Larval chaetae occur only in notopodia. Pygidium large and round, acquires dorsal cirri in late larvae. Late larvae have two antero-lateral processes on prostomium (Fig. 4H). Pigmentation usually absent, some individuals have brownish pigmentation on peristomium, dorsum, and/or pygidium, and/or two medial black pigmentation ventrally on approximately chaetiger VI and anterior margin of the pygidium.
This species is recorded from Japan for the first time in the present study. The brooding of larvae beneath dorsal branchiae in this species was observed in September 2011 (Fig. 6). The larvae adhere to their parents and are enclosed by branchiae present on the posterior chaetigers (26 th -39 th chaetigers). The larvae are retained on the parents' dorsum even when the parent individuals leave their tube, unless the parent is disturbed. Larvae seemed to be released at around the 3-chaetiger stage; the fact that planktonic larvae with more than three chaetigers were commonly collected from plankton supports this observation. Similar dorsal larval brooding was reported in other Rhynchospio species (Levin 1982;Radashevsky 2007;Radashevsky et al. 2014) and in Streblospio benedicti Webster, 1879 (Levin 1982(Levin , 1984. Planktonic larvae were found in Onagawa Bay, Gamo Lagoon, and Sasuhama in almost every season of the study period, but few were found in winter season (November to March). Larval morphology of this species resembles that of R. glutaea and R. nhatrangi Radashevsky, 2007described by Carrasco (1976 and Radashevsky (2007), respectively. The overall larval morphology of Rhynchospio species is quite similar to that of the genus Malacoceros described in Hannerz (1956, as Scolelepis), but it differs in the latter having three pairs of black eyes, the most lateral pairs with double-eyes.
Identification key to species of the larvae belonging to the genus Scolelepis in northeastern Japan Larval morphology. Thick and fusiform in shape. Prostomium pointed anteriorly, terminates in retractile, muscular tip. Lateral parts of peristomium clearly demarcated from prostomium, forming peristomial umbrella. Peristomial umbrella carrying well-developed prototroch. Two pairs of red eyes arranged in somewhat trapezoidal shape, medial pair situated anteriorly. Greenish pigment in pharynx and proctodaeum. Pygidium very broad and horseshoe-shaped. Remarks. Only three individuals of early larvae of this species were collected from Sasuhama in January 2012. The 18S rRNA gene sequences obtained in the present study for these specimens match (464/464 bp) that of S. kudenovi from Lizard Island, Australia (KP636517: Meißner and Götting 2015). Since the species identification is unreliable because of the short reference sequence, this species was referred to S. cf. kudenovi.
The sequence of an adult individual, which collected from the surf zone of the sandy beach in Rishiri Island and previously identifies as Scolelepis kudenovi (Abe et al. 2019c) as the morphology agrees with the descriptions of S. kudenovi by Imajima (1992) and Meißner and Götting (2015), 100% matched with that of the larvae of Scolelepis cf. kudenovi in 18S rRNA gene (1819/1819 bp) but largely differed in 16S rRNA gene (462/505 bp). Because the 16S rRNA gene of the adult individual was rather closer to S. daphoinos (430/455 bp) from China (GU362676, Zhou et al. 2010), it is referred to S. aff. daphoinos in the present study (Table 1, Figs 2, 3). Fig. 4J, K Larval morphology. Thick and fusiform in overall shape. Prostomium pointed anteriorly as a small process, tip of prostomium terminates in retractile, muscular tip. Lateral parts of peristomium clearly demarcated from prostomium, forming large peristomial umbrella. Peristomial umbrella carrying well-developed prototroch. Short palps developed in late larvae, attached on lateral-most parts of peristomium. Two pairs of red eyes arranged in an approximately straight line. Pharynx pigmented orange and the gut pigmented brown. Pygidium broad and spherical.

Scolelepis sp. 1
Remarks. Adult individuals of this species were collected from muddy sediments at 22 m depth in Onagawa Bay in December 2011 by using a Smith-McIntyre grab sampler. These adults were morphologically identified as Scolelepis, but they were not identified to species level as these specimens were all incomplete and in poor condition. As the 18S and 16S rRNA gene sequences obtained in the present study did not match any available Scolelepis sequences (Figs 2, 3), this species was referred to Scolelepis sp. 1. Planktonic larvae of this species were collected in Onagawa Bay in October during the study period. The larvae and adults were confirmed to match using molecular data (Fig. 3). Fig. 4L Larval morphology. Thick and fusiform in overall shape. Prostomium bluntly pointed anteriorly, terminates in retractile, muscular tip. Lateral parts of peristomium clearly demarcated from prostomium, forming large peristomial umbrella. Peristomial umbrella carrying well-developed prototroch. Short palps developed in late larvae, attached on lateral-most parts of peristomium. Two pairs of red eyes arranged in an approximately straight line. Pharynx widely pigmented orange and the gut pigmented brown. Pygidium broad and spherical shaped.

Scolelepis sp. 2
Remarks. No adult individuals of this species were collected in the present study. Even though the 18S and 16S rRNA gene sequences obtained from larvae in the present study did not match any available Scolelepis sequences, this species was referred to Scolelepis sp. 2 as the larvae constitute a monophyletic clade with the other Scolelepis species with robust statistical support (Figs 2, 3).
Planktonic larvae of this species were found in Onagawa Bay in September. The larvae of this species are quite similar to those of Scolelepis sp. 1; however, the prostomium is more broadly pointed anteriorly and the gut diverticula are more strongly segmented in the former species.

Genus Spiophanes Grube, 1860
Larval diagnosis. Overall shape slender. Prostomium small or broad, rounded or slightly notched anteriorly. Lateral parts of the peristomium slightly or moderately demarcated from the prostomium, palps on lateral-most parts of peristomium. Two pairs of red eyes present. In late larvae, a pair of prominent or small antero-lateral processes on prosto-mium are often developed. Melanophore absent, some pigmentation patches of various colors are present on pharynx, proctodaeum, prostomium, peristomium, pygidium, and/ or various locations of the body in late larvae. Nototrochs occur in all chaetigers from chaetigers II-IV onwards. Gastrotrochs occur in all chaetigers from chaetiger II onwards (Thorson 1946;Hannerz 1956;Carrasco 1976;Plate and Husemann 1994;Blake 2006).
Identification key to species of the larvae belonging to the genus Spiophanes in northeastern Japan Larval morphology. Overall shape slender. Prostomium small and rounded anteriorly. Two pairs of red or dark red eyes present, lateral pair situated anteriorly. Late larvae bear very small antero-lateral processes on prostomium. Lateral parts of peristomium slightly demarcated from prostomium, palps attached on lateral-most parts of peristomium. Nototrochs occur from chaetiger IV onwards (Fig. 4N). Yellow pigments on the prostomium and pygidium, intense yellow-brown pigment on peristomium, inside of pharynx, and pygidium. Small red pigment spots present on lateral part of body (Fig. 4N). Black pigment in pharynx and proctodaeum absent.
Only a few larvae of this species were collected in Onagawa Bay in November 2011. The overall larval morphology of this species somewhat resembles that of S. kroyeri de-scribed by Hannerz (1956) in the following aspects: prostomium is relatively small and anteriorly rounded, the peristomium is not quite sharply demarcated from the prostomium, nototrochs occur from chaetiger IV onwards, and the brown pigmentation is present on the pygidium and inside the pharynx. However, S. kroyeri lacks small red pigment spots on the lateral part of the body and lateral processes on the prostomium even in 22-chaetiger larvae.
Spiophanes aff. uschakowi Zachs, 1933 Fig. 4O, P Larval morphology. Overall shape slender. Prostomium broad and slightly notched anteriorly. In late larvae, a pair of prominent antero-lateral processes on prostomium developed. Two pairs of red or dark red eyes present, lateral ones situated somewhat anteriorly. Lateral parts of peristomium moderately demarcated from prostomium, palps on lateral-most parts of peristomium. Nototrochs occur from chaetiger II onwards. Pharynx and proctodaeum black in color internally. Pygidium acquires dorsal cirri in late larvae. Some brownish, yellowish, or greenish pigmentation occurred on various locations of body in late larvae.
Remarks. Adult individuals of this species were collected from muddy bottom sediments at 22 m depth in Onagawa Bay in April and May 2012 by using a Smith-McIntyre grab sampler and from bottom sediments of the shallow subtidal zone in Sasuhama in February 2012. Adult morphology agrees with the description of S. cf. uschakowi by Meißner and Blank (2009) as well as with that of S. bombyx by Imajima (1991b). However, the 18S and 16S rRNA gene sequences of this species did not match those of S. uschakowi obtained from DDBJ/EMBL/GenBank (KM998760): there was a 0.29% (5/1750 bp) and 0.88% (3/342) difference, respectively between these two species. Therefore, this species was referred to S. aff. uschakowi. The larvae and adults were confirmed to 100% match using molecular data (Fig. 2).
Planktonic larvae of this species were collected in Onagawa Bay in November 2011 and in Sasuhama in February 2012. The larval morphology of S. aff. uschakowi was different from that of S. uschakowi in the following aspects: prostomium of the former is broad and slightly notched anteriorly, whereas that of the latter is relatively small and anteriorly rounded; the peristomium of the former is well demarcated from the prostomium, but that of the latter is relatively less demarcated; nototrochs of the former occur from chaetiger II onwards, whereas those of the latter occur from chaetiger IV onwards; pigmentation inside the pharynx is black in the former but brown in the latter; and black pigmentation in the proctodaeum is present in the former but absent in the latter. Black pigmentation in the pharynx and proctodaeum were also reported in the larvae of S. bombyx (Hannerz 1956), S. cf. bombyx (Blake 2006), and S. duplex (Chamberlin, 1919) (Blake 2006). However, the illustrations of S. cf. bombyx provided by Blake (2006: fig. 13.10C, D) are seemingly more similar to larvae of Rhynchospio than to those of Spiophanes.
Identification key to species of the larvae belonging to the genus Boccardia in northeastern Japan Larval morphology. Slender and fusiform in overall shape, widest in middle of body. Prostomium rounded and slightly notched anteriorly. Three pairs of eyes present, most median pair rounded, lateral pairs double-eyes. Body entirely faint green in color. A prominent row of dorsal melanophores occurs medially from chaetiger III, lateral black pigment spots present on chaetigers VII and VIII in late larvae (Fig. 7B). Pygidium has dorsal gap, pigmented with weak dark color. Internally, vestibule light brown, gut either yellow or brown. Gastrotrochs on chaetigers V and VII.
Planktonic larvae of this species were rare, and only one 15-chaetiger larva (Fig. 7B) was collected in Sasuhama in May 2011. Another 9-chaetiger larvae, which accidentally hatched from its egg capsule in an adult tube during the process of extraction of the adult specimens (Fig. 7A), was also collected on the same date. Boccardia proboscidea has been reported to have poecilogonous development (Gibson 1997;Oyarzun et al. 2011). However, Sato-Okoshi (2000) reported that Japanese populations only show lecithotrophic development, with no (or a very short) planktonic stage after hatching. The larval morphology of this species agrees with the description of that of B. proboscidea documented in Hartman (1941), Woodwick (1977), Blake and Kudenov (1981), Gibson (1997), Gibson and Smith (2004), Kamel et al. (2010), and Oyarzun and Brante (2015). The dorsal pigment pattern of these larvae resembles that of the larvae of B. tricuspa (Hartman, 1939) described by Carrasco (1976, as B. proboscidea;fide Blake and Kudenov 1978), B. natrix (Söderström, 1920) described by Söderström (1920, as Polydora natrix), and B. columbiana Berkeley, 1927 described by Blake and Arnofsky (1999) and Blake (2006) in having a single row of mid-dorsal melanophores. However, the dorsal pigment pattern of the larvae of B. tricuspa differs from that of B. proboscidea in having branching dorsal melanophores on chaetiger I and in lacking small black lateral pigment spots on chaetigers VII and VIII. The larvae of B. natrix also lack small black lateral pigment spots on chaetigers VII and VIII. Boccardia columbiana has extensively branching mid-dorsal melanophores from chaetiger II onward, whereas middorsal melanophores are less branching and start from chaetiger III in B. proboscidea. Fig. 7C Larval morphology. Slender and fusiform in overall body shape. Prostomium rounded with a slight anterior notch. Three pairs of eyes present, most median pair rounded and lateral pairs double-eyes. Body entirely faint yellow. Dorsal melanophore absent, slight black pigmentation present (Fig. 7C, left). Pygidium with dorsal gap, pigmented with yellow. Internally, the vestibule and pharynx brown or black, gut green in color. Gastrotrochs on chaetigers V and VII.

Boccardia pseudonatrix Day, 1961
Remarks. Adults of this species were non-boring and collected from mud deposits in crevices of shells of living C. gigas oysters in Tomiura. Adult morphology (see Abe et al. 2019b) agrees with the descriptions of B. pseudonatrix from South Africa (Day 1967;Simon et al. 2010) and Australia (Sato-Okoshi et al. 2008, as B. knoxi;see Walker 2013). The 18S rRNA gene sequences obtained in the present study completely match (1714/1714 bp) that of B. pseudonatrix from South Africa (KY677895) reported by Williams et al. (2017) (Fig. 3). Therefore, this species was referred to B. pseudonatrix.
Boccardia pseudonatrix has been reported to have adelphophagic larvae with a short or absent planktonic phase (Sato-Okoshi et al. 2008;Simon 2015). The larvae herein reported accidentally hatched from egg capsules in an adult tube during the process of extraction of the adult specimens.
Boccardia sp. 1 Fig. 7D-F Larval morphology. Slender and slightly fusiform in overall shape, widest in anterior part of body. Prostomium rounded anteriorly. Three pairs of black eyes present, most median pair rounded and lateral pairs double-eyes. Body entirely yellowish in color. A prominent row of dorsal melanophores occurs medially from chaetiger IV, lateral black pigment spots present from chaetiger V onwards (Fig. 7E). Pygidium with dorsal gap, pigmented with black color. Vestibule black, gut orange in color internally. Larval chaetae on first chaetiger long especially in early larvae (Fig. 7D). Gastrotrochs on chaetigers III, V, VII, X, and XIII.
Remarks. No adult individuals of this species were collected in the present study. The 18S and 16S rRNA gene sequences obtained from the larvae did not match any available Boccardia sequences, but this species is very similar to the other Boccardia species in larval morphology and gene sequences (Figs 2, 3). Therefore, this species was referred to Boccardia sp. 1.
Planktonic larvae of this species were collected from Onagawa Bay in April, May, November, and December 2011, and January, February, March, and May 2012. The overall body shape of these larvae is slender and slightly fusiform, similar to those of B. proboscidea. However, other larval morphological characteristics differ between these two species: overall body color is faint yellow in the former species and faint green in the latter; the larval chaetae are longer in the former species than in the latter, especially in early larvae (Fig. 7D); lateral black spots are present on chaetiger V onwards in the former species but only on chaetigers VII and VIII in the latter species.
Boccardia sp. 2 Fig. 7G Larval morphology. Thick and fusiform in overall shape, widest at middle part of body. Prostomium extensively broad and anteriorly rounded. Three pairs of black eyes present, most median pair rounded and lateral pairs double-eyes. Body entirely faint green in color in late larvae. A prominent row of dorsal ramified melanophores occurs medially from chaetiger IV onwards, lateral black pigment spots absent. Pygidium with dorsal gap, pigmented with weak dark color. Internally, vestibule black, gut orange in color. Gastrotrochs on chaetigers III, V, VII, X, and XIII.
Remarks. No adult individuals of this species were collected in the present study. The 18S and 16S rRNA gene sequences herein obtained from the larvae did not match any of the available Boccardia sequences, but this species is similar to the other Boccardia species in larval morphology and gene sequences (Figs 2, 3); therefore, this species was referred to Boccardia sp. 2.
Planktonic larvae of this species were collected from Onagawa Bay in December 2010 and November and December 2011, from Sasuhama in January 2013, and from Sendai Port in December 2010. The larval morphology of this species differs from that of other Boccardia larvae in having a thick and fusiform body shape.

Genus Boccardiella Blake & Kudenov, 1978
Larval diagnosis. Overall shape thick and fusiform. Prostomium extensively broad and rounded anteriorly. Three pairs of black eyes present, most lateral pairs usually double-eyes. More than two pairs of dorsal melanophores from chaetiger III onwards. Lateral and ventral pigments present. Nototrochs occur in all chaetigers except first two. Gastrotrochs occur in irregular pattern. Modified chaetae develop in chaetiger V in late larvae (Rullier 1960, as Polydora redeki;Dean and Blake 1966, as Boccardia). Fig. 7H, I Larval morphology. Thick and fusiform in overall shape, widest at middle part of body. Prostomium broad and anteriorly rounded, usually dusky brown anteriorly. Three pairs of black eyes present, most median pair rounded, lateral pairs usually double-eyes, occasionally divided into respective eyes. Black pigmentation usually presents ventrally on each lateral lip, occasionally absent. Dorsal pigmentation basically consists of a pair of medial bands, lateral branching melanophores, and small pigment patch at the base of notopodia in each chaetiger from chaetiger III onwards (Fig. 7H). These melanophores undergo expansion and contraction, sometimes coalescing to cover almost the whole of the dorsal surface as ramified pigmentation (Fig. 7I). Four transverse lines of black pigmentation sometimes fused as a single transverse band in chaetiger I. One or two pairs of lateral black pigmentation on chaetiger II. Two rows of band-shaped ventral pigmentation usually located on posterior edges of some chaetigers posterior to second chaetiger. A pair of black pigment patches on pygidium. Gastrotrochs on chaetigers III, V, VII, X, and XIII.

Boccardiella hamata (Webster, 1879)
Remarks. Adults of this species were non-boring and collected from mud deposits in crevices of shells of living C. gigas oysters in Sasuhama in May 2011 and February 2016. Adult morphology agrees with the description of B. hamata by Sato-Okoshi (2000). Therefore, this species was referred to B. hamata. The larvae and adults were confirmed to match (18S: 1772/1772, 16S: 480/481 bp) using molecular data (Fig. 2).
Planktonic larvae of this species were frequently collected from Onagawa Bay, Gobu-ura, and Sasuhama in July and August. The larval morphology of this species agrees with that of B. hamata described by Dean and Blake (1966, as Boccardia).

Genus Dipolydora Verrill, 1879
Larval diagnosis. Overall shape slender or slightly fusiform. Prostomium small rounded anteriorly. Three pairs of black eyes present, most lateral pairs often double-eyes. Ramified melanophore between central and lateral pairs of eyes usually absent, but present in some species (e.g., D. cf. commensalis). Dorsal pigment pattern consists of two rows of band or spot shaped melanophores or a transverse row of small melanophores at each chaetiger in most species, while some species have single row of branching mid-dorsal melanophores (e.g., D. cf. commensalis) or completely lack melanophores (e.g., D. armata). Lateral and ventral pigments are present or absent. Nototrochs occur in all chaetigers except the first two chaetigers. Gastrotrochs occur in irregular pattern. Modified chaetae develop in chaetiger V in late larvae (Andrews 1891, as Polydora;Hannerz 1956, as Polydora;Hatfield 1965, as Polydora;Blake 1969, as Polydora;Carrasco 1976, as Polydora;Day and Blake 1979, as Polydora;Radashevsky 1989, as Polydora;Plate and Husemann 1994, as Polydora;Lewis 1998;Blake 2006;Blake 2017).
Identification key to species of the larvae belonging to the genus Dipolydora in northeastern Japan Remarks. Adults of this species are shell-borers and were collected from shells of wild C. gigas oysters in Sasuhama in July 2012. Adult morphology agrees with the description of D. bidentata by Sato-Okoshi (1999). The 18S and 16S rRNA gene sequences obtained in the present study match (18S: 900/900, 16S: 473/475 bp) that of D. bidentata from Russia (JX228065) reported by Radashevsky and Pankova (2013) (Fig. 3). Planktonic larvae of this species were collected from Onagawa Bay in Novem-ber 2011 and from Sasuhama in February 2012. The larvae and adults were confirmed to 100% match using molecular data (Fig. 2). Dipolydora cf. commensalis (Andrews, 1891) Fig. 7L Larval morphology. Overall shape elongated and slender. Prostomium small but wider than body and rounded anteriorly. Three pairs of eyes present, most lateral pairs double-eyes of kidney-shaped appearance. Ramified melanophores present around eyes. Black pigmentation on lateral peristomium absent. Median row of ramified melanophores from chaetiger I onwards. Lateral and ventral pigments absent. A central black pigment spot and a pair of dark brown pigments on pygidium. Pygidium has a dorsal notch and lacks appendages. Gastrotrochs on chaetigers III, V, VII, X, XIII, XV, XVII, XIX, XXI, and XXIII. Modified chaetae develop in chaetiger V in late larvae.
Remarks. No adults of this species were collected in the present study. The 18S and 16S rRNA gene sequences obtained from the larvae of this species neither match nor constitute a monophyletic clade with any of the other available spionid sequences (Figs 2, 3). However, this species was tentatively identified as D. cf. commensalis based on its larval morphology, as it includes the characteristic dorsal pigment pattern of larvae of D. commensalis as described by Andrews (1891), Hatfield (1965), Blake (1969), and Radashevsky (1989) (described as Polydora commensalis by all of these authors). The combination of a slender body and a single dorsal median row of distinct melanophores from chaetiger I to the end of the body is distinctive among spionid larvae and has not been reported for any other spionid species. Currently, there are no records of D. commensalis from Japan; however, the presence of this species in Japan is expected as it has been reported from the Asian continental coast of the Sea of Japan and the Kurile Islands (Radashevsky 1993). This species is an obligate symbiont of hermit crabs (Blake 1996;Williams and McDermott 1997), but little effort was devoted to collecting hermit crab shells in the present study.
Notably, the results of the phylogenetic analysis in the present study showed that D. cf. commensalis deviates from the monophyletic clade constituted by many other Dipolydora species. This result supports the suggestion by Blake (1971) that D. commensalis may represent a distinct genus as its morphology deviates widely from other species of the genus Polydora and Dipolydora.
Only three individuals of planktonic larvae of this species were collected in Sasuhama in January 2013. A small patch of lateral black pigments on the anterior margin of each chaetiger in late larvae was described in Hatfield (1965) and Blake (1969). However, these pigments were not observed in the present study, as in Andrews (1891) and Radashevsky (1989). Although Hatfield (1965) and Blake (1966) noted the high similarity between the larval morphologies of D. commensalis and Polydora hermaphroditica Hannerz, 1956, the adult morphologies of these two species were reported to be completely different (Bhaud 1966). The dorsal pigment pattern of P. hermaphroditica larvae reported by Hannerz (1956) rather resembles those of Polydora glycymerica and Polydora cf. glymymerica larvae reported by Radashevsky (1989) and in the present study, respectively. Fig. 7M, N Larval morphology. Overall body shape elongated and slender. Prostomium small and rounded anteriorly. Three pairs of black eyes arranged in transverse row, most lateral pairs double-eyes. Black pigmentation on lateral peristomium absent. Two pairs of dorsal black spots begin on chaetiger III onwards and continue to posterior end, sometime medial pair in first 2-4 chaetigers band-shaped. Small medial spot of black pigment on posterior margin of each chaetiger usually from chaetigers III, rarely from V or VI, in late larvae. Two small spots of black pigmentation occur lateral to the medial black pigmentation from approximately chaetiger VI or VII. A small black pigment spot, not visible dorsally, present on antero-lateral edges from chaetiger II onwards. Black pigment occurs on pygidium. Rust-colored pigment occurs in pharynx. Ventral pigment absent. Some metamorphosing larvae reduce pigmentation over the entire body and present whitish appearance with eyes fused and appears as one pair (Fig. 7N). Gastrotrochs on chaetigers III, V, VII, X, XIII, and XV.

Dipolydora giardi (Mesnil, 1896)
Remarks. Adults of this species are shell-borers and were collected from shells of cultured Mizuhopecten yessoensis (Jay, 1857) (formerly as Patinopecten yessoensis) scallops suspended in Onagawa Bay in December 2010. Adult morphology agrees with the description of D. giardi by Sato-Okoshi (1999). Therefore, this species was referred to D. giardi. The larvae and adults were confirmed to match using molecular data (Fig. 3).
The 18S rRNA gene sequences of this species are very similar to that of D. capensis 1PE from South Africa (KY677896) reported by Williams et al. (2017), but there is a slight difference between their sequences (0.12% difference: 2/1714 bp). It is unclear whether this difference indicates that these two are the same or different species because two different 18S rRNA gene sequences have been reported from South Africa and are currently under the same species name (D. capensis) (Table 2, Fig. 3). No gene sequences of D. cf. giardi previously recorded from South Africa (Simon 2011) are available.
Planktonic larvae of this species were collected from Onagawa Bay in December 2010, June, July, October, November, and December 2011, and December 2012, and from Sasuhama in January 2013. The larval morphology of this species was previously described from California by Day and Blake (1979, as Polydora giardi). The morphology and dorsal pigment pattern of late larvae described by these authors resembles that reported here, but there are slight differences: two golden pigment spots present on either side of chaetiger I in the former description but absent in the latter; two small lateral melanophores present on chaetigers I and II in the former description but absent in the latter; a medial black pigmentation beginning from chaetiger II onwards in the former description but from chaetiger III onwards in the latter description; and two small spots of black pigmentation lateral to the medial black pigmentation starting from chaetiger III in the former description but from more posteriorly in the latter description. These differences between specimens from Japan and California may indicate that they are different species, or that intraspecific variation occurs in larval dorsal pigmentation. Day and Blake (1979) pointed out differences in reproductive traits between the Californian and French populations and suggested the existence of two different species. Therefore, more than one species may be included under the name of D. giardi, which currently is reported with a worldwide distribution (Radashevsky and Petersen 2005). Fig. 7O Larval morphology. Late larvae usually thick and slightly fusiform in shape, although not as much as the larvae of Boccardia sp. 2 (Fig. 7G), Boccardiella hamata (Fig. 7H, I), and Pseudopolydora species (Fig. 9A-I). Anterior margin of prostomium has yellowbrown pigment. Three pairs of black eyes arranged in transverse row, most lateral pairs double-eyes. Band of black pigment on each lateral part of the peristomium. First dorsal black melanophores occur as paired bands on chaetiger III and continue through to chaetiger V. From chaetiger VI, two pairs of dorsal black spots or bands occur and continue to posterior end of body. From chaetiger IV or V and continuing posteriorly, clusters of small black pigmented cells present in transverse row on dorsal posterior half of chaetigers. Lateral pigment found on late larvae on chaetiger II. Each notopodial lobe tipped with orange pigment, small patch of black pigment at the base of notopodial lobes. Ventral pigment consists of paired bars on posterior border of chaetigers, commencing with chaetiger II. Some black or brown pigment may occur on pygidium. Gastrotrochs occur on chaetigers III, V, VII, X, XIII, XV and XVII.

Dipolydora cf. socialis (Schmarda, 1861)
Remarks. Adults of this species were non-boring and collected from muddy bottom sediment at 22 m depth in Onagawa Bay in December 2010 by using a Smith-McIntyre grab sampler and from bottom sediments of shallow subtidal zone in Sasuhama in April 2013. Adult morphology agrees with the description of D. socialis by Sato-Okoshi (2000). The 18S rRNA gene sequence obtained in the present study showed a 0.35% (6/1715 bp) difference with that of D. cf. socialis from South Africa (KY677899) reported by Williams et al. (2017), which may indicate that these two are different species. The 18S rRNA gene sequence obtained in the present study rather closer to that of D. carunculata (940/942 bp match) reported by Radashevsky and Pankova (2013), but the 16S gene sequence showed a 2.3% (11/475 bp) difference with that of D. carunculata. As described above, since the taxonomic status of the species reported here is uncertain, we tentatively referred to it as D. cf. socialis.
Planktonic larvae of this species were collected from Onagawa Bay in November 2010 and 2011, and in October 2012. The larvae and adults were confirmed to match (18S: 1770/1770, 16S: 473/475 bp) using molecular data (Fig. 2). The larval morphology of this species agrees with that of D. socialis described as Polydora socialis by Blake (1969) and Carrasco (1976). Dipolydora sp. Fig. 7P Larval morphology. Overall body shape slender. Prostomium small and rounded anteriorly. Anterior margin of prostomium has weak yellow-brown pigment. Three pairs of black eyes present in transverse row, most lateral pairs double-eyes. Black pigmentation on lateral peristomium absent. A pair of dorsal black spots present on chaetiger III onwards. A small medial spot of black pigment on posterior margin of chaetiger III. Some black pigment occurs on pygidium. Ventral pigment absent. Gastrotrochs occur on chaetigers III and V.
Remarks. No adult individuals of this species were collected in the present study. The 18S and 16S rRNA gene sequences obtained from larvae in the present study did not match any available Dipolydora sequences. As the larvae specimens formed a monophyletic clade with the other Dipolydora species (excluding D. armata,D. capensis 1GG,D. cf. commensalis,and D. quadrilobata) with robust statistical supports (Figs 2, 3), this species was referred to Dipolydora sp.
Identification key to species of the larvae belonging to the genus Polydora in northeastern Japan Larval morphology. Overall shape slender and slightly fusiform. Prostomium broad and rounded anteriorly. Three pairs of black eyes present, innermost pair rounded, lateral pairs double-eyes, ramified melanophore between innermost and lateral two pairs of eyes usually present. Black pigment on lateral peristomium absent. Dorsal pigmentation consists of two rows of melanophores from chaetiger III. Dorsal melanophores undergo expansion and contraction, may expand to branching melanophores or ramified appearance or covered almost whole of dorsal surface by very finely ramified black pigments (Fig. 8B), or they contract to dot-like pigmentation patches (Fig. 8A). Lateral and ventral pigments absent. Vestibule and pharynx pigmented with black, gut pigmented with orange color. Modified chaetae develop on chaetiger V in late larvae. Gastrotrochs occur on chaetigers III, V, VII, X, XIII, XV, and XVII.
Remarks. Adults of this species are boring and were collected from shells of cultured M. yessoensis scallops suspended in Onagawa Bay in February 2011 and Mutsu Bay in October 2011. This species was identified as P. brevipalpa as adult morphology agrees with the descriptions by Sato-Okoshi (1999) and Sato-Okoshi and Abe (2012). The larvae and adults were confirmed to 100% match using molecular data (Fig. 2).
Planktonic larvae of this species were collected from Onagawa Bay in April, May, and July 2011 and from Sasuhama in April 2011. The pair of large ramified or dot-like melanophores from chaetiger III distinguishes larvae of this species from those of other Polydora species. Blake (2017) reported similar dorsal pigment patterns in the larvae of Polydora spongicola Berkeley & Berkeley, 1950. However, the larvae of P. spongicola in Blake (2017) differ from those of P. brevipalpa in having dorsal melanophores from chaetiger II instead of chaetiger III, dark green colored intestine instead of orange, and non-pigmented pharynx instead of pigmented with black. Reproduction and life history of this species was reported in Sato-Okoshi et al. (1990) and Sato-Okoshi (1994) (both as P. variegata). Fig. 8C, D Larval morphology. Overall shape slender. Prostomium broad and rounded anteriorly. Three pairs of black eyes present, median pair rounded, most lateral pairs doubleeyes, ramified melanophores between first median and the second lateral pair of eyes usually present. In late larval stage, anterior part of prostomium and lateral lips of peristomium pigmented yellow or brown. Small spots of black pigments occur on lateral parts of peristomium. Dorsal pigmentation consists of two rows of melanophores from chaetiger III with those of anterior four chaetigers band-shaped and then replaced by rounded or ramified melanophores from chaetiger VII onwards. Three rows of small faint dorsal spots of brown pigment present on posterior edge from chaetigers III or IV onwards in late larvae. Lateral pigment on chaetigers II, III, and often VI-XI extensive compared to that on other chaetigers. Large yellow or brown chromatophores occur ventrally from chaetigers V or VI onwards, usually three chromatophores arranged in transverse line except on gastrotroch-bearing chaetigers where single midventral chromatophores present. Black pigment spots occur on ventral side of body (Fig. 8D) and mid-dorsal part on pygidium (Fig. 8C). Gastrotrochs occur on chaetigers III, V, VII, X, XIII, XV and XVII.

Polydora cornuta Bosc, 1802
Remarks. Adults of this species were non-boring and collected from mud deposits in crevices of shells of living C. gigas oysters in Sasuhama in June 2011 and from intertidal bottom sediment in Gamo Lagoon in August 2012. This species was identified as P. cornuta as adult morphology agrees with the description by Sato-Okoshi (2000) and Radashevsky (2005). The larvae and adults were confirmed to match (18S: 1770/1770, 16S: 468/470 bp) using molecular data (Fig. 2). Rice et al. (2008) suggested that at least three sibling species may be involved in North America under the name of P. cornuta by differences of mitochondrial COI sequences between California, Florida, and Maine populations. Takata et al. (2011) reported that the P. cornuta from Fukuyama in the Seto Inland Sea, western Japan is genetically close with the California/New Zealand lineage. It is unclear to which lineage the eastern Japan populations belong. The 18S rRNA gene sequence obtained in the present study showed a 1.9% (5/421 bp) difference with that of P. cornuta from Netherlands (KC686637).
Planktonic larvae of this species were collected from Gamo Lagoon in August 2012. The larval morphology of this species generally agrees with the descriptions of P. cornuta by Hannerz (1956, as P. ligni), Blake (1969, as P. ligni), Plate andHusemann (1994, as P. ligni), andRadashevsky (2005). Peristomial melanophores, which were reported by Hannerz (1956) and Blake (1969) but not by Radashevsky (2005), and middorsal vesiculate melanophores, which were reported by Radashevsky (2005) but not described by Hannerz (1956) and Blake (1969), were both present in specimens of the present study. Ventral pigmentation pattern was consistent with the description by Blake (1969) and Radashevsky (2005) instead of Hannerz's (1956) description. The larval dorsal pigmentation pattern, similar to that of P. cornuta, is typically found in many other Polydora species. This species can, however, be distinguished by the characteristic ventral yellow pigmentation pattern as the yellow pigment on the ventral side of the other Polydora species is diffusely scattered and does not appear regularly arranged when present (Radashevsky 2005).
Polydora cf. glycymerica Radashevsky, 1993 Fig. 8E Larval morphology. Overall shape elongated and slender. Prostomium small and rounded anteriorly. Three pairs of black eyes present, most lateral pairs double-eyes. Ramified melanophores between middle and lateral pair of eyes absent. Pigmentation on lateral peristomium absent. Two rows of ramified melanophores on chaetigers III-VI, and a median row of ramified melanophores from chaetiger VII onwards. Lateral and ventral pigments absent. A pair of black pigments occur on pygidium. Pygidium has a dorsal notch and lacks appendages. Gastrotrochs absent in 25-chaetiger larvae, probably already lost. Modified chaetae develop on chaetiger V.
Remarks. No adult individuals of this species were collected in the present study. The 18S and 16S rRNA gene sequences obtained from larvae in the present study did not match any of the available Polydora sequences. However, as the larvae formed a robustly supported clade with other Polydora species (Figs 2, 3), this species was referred to as the genus Polydora. Furthermore, the larval morphology including the characteristic dorsal pigment pattern of this larvae matches that of the larvae of P. glycymerica described by Radashevsky (1989). Therefore, this larva was tentatively identified as P. cf. glycymerica. However, there were slight differences between the present specimens and Radashevsky (1989) description: the two rows of ramified melanophores continued until chaetiger VI in the present description, whereas it continues to chaetigers VII-X according to Radashevsky (1989); ramified melanophores between the middle and lateral pair of eyes are present in the former description while absent in the latter; larvae of P. cf. glycymerica collected in the present study were 25-chaetigers with > 2.0 mm long (Fig. 8E), whereas the largest larva observed by Radashevsky (1989) was a 20-chaetiger specimen 1.8 mm long. Further studies should test whether these differences are attributable to individual or developmental variabilities or interspecific differences.
The dorsal median single row of ramified melanophores is distinct in the larvae of the genus Polydora. The larvae of Polydora hermaphroditica also have a dorsal median row of ramified melanophores such as that of the larvae of P. cf. glycymerica and P. glycymerica (Hannerz 1956;Plate and Husemann 1994). However, the first species differs from the other two by the absence of two rows of ramified melanophores on anterior chaetigers.
Only one individual of planktonic larva of P. cf. glycymerica was collected in Onagawa Bay in October 2011. Polydora glycymerica was previously recorded as a shell-borer of Macridiscus aequilatera (G. B. Sowerby I, 1825) from Oarai, Japan (Sato-Okoshi 1999). Fig. 8F Larval morphology. Overall body shape slender or somewhat fusiform. Prostomium broad and rounded anteriorly. Three pairs of black eyes present, most lateral pairs double-eyes. Ramified melanophores between first and second innermost pair of eyes absent. Black pigmentation patches on lateral peristomium absent. Dorsal pigmentation consists of two rows of melanophores from chaetiger III with those of first five pairs band-shaped and then replaced by ramified melanophores in posterior chaetigers. Lateral pigments found on late larvae on chaetigers II-IV. Dorsolateral pigments at base of the parapodia start from chaetiger VII. A pair of black pigment occur on pygidium. Ventral pigment absent. Modified chaetae develop in chaetiger V in late larvae. Gastrotrochs occur on chaetigers III, V, VII, X, XIII, and XV.
Only late larvae were found in July in Onagawa Bay. The larval morphology of this species agrees with descriptions by Wilson (1928) and Radashevsky and Migotto (2017). This species has adelphophagic and lecithotrophic larval development, in which larvae feed on nurse eggs in brood capsules, hatch at a very late stage, and have only a short pelagic life (Wilson 1928;Read 1975;Sato-Okoshi et al. 2008, as P. uncinata;Radashevsky and Migotto 2017). The poecilogenous development of this species with planktotrophic and adelphophagic planktonic larvae was reported by , , and Simon (2015). Fig. 8G-I Larval morphology. Overall body shape slender. Prostomium slightly broad and rounded anteriorly. Three pairs of black eyes present; median pair of eyes rounded, most lateral pairs double-eyes. Ramified melanophore between middle and lateral pair of eyes usually present (Fig. 8G, H). Weak brown pigmentation located on lateral parts of peristomium, behind prototroch, occasionally much paler or absent. Dorsal pigmentation consists of two rows of melanophores from chaetiger III with those of first IV-VI band-shaped and subsequently replaced by ramified melanophores. These melanophores undergo expansion and contraction. Lateral pigment found on chaetigers II and III in late larvae (Fig. 3G). Dorsolateral pigment at base of most parapodia, often appears to coalesce with dorsal pigment bands on anterior part of body (Fig. 8H). Pygidium has a dorsal notch and lacks appendages; a pair of black pigment patches occur on pygidium. Ventral brown pigment may be present on posterior part of late larvae (Fig. 8I). Telotroch well developed. Gastrotrochs on chaetigers III, V, VII, X, XIII, and XV; those of chaetigers III and V lost in late larvae (Fig. 8I). In late larvae, modified chaetae develop in chaetiger V.

Polydora onagawaensis Teramoto, Sato-Okoshi, Abe, Nishitani & Endo, 2013
Remarks. This species is a shell-borer, and adult individuals were collected from shells of the wild turban snail O. rusticus, cultured scallop M. yessoensis, and wild and cultured C. gigas oysters in Onagawa Bay and Sasuhama, northeastern Japan. This species was identified as P. onagawaensis as adult morphology agrees with the description by Teramoto et al. (2013). The larvae and adults were confirmed to match (18S: 1771/1771, 16S: 472/473 bp) using molecular data (Fig. 2).
Planktonic larvae of this species were abundant from November to June in Onagawa Bay during the study period. The larval morphology of this species is similar to that of Polydora sp. 3 (see below). However, the former species has weak brown pigmentation on the lateral parts of the peristomium, whereas the latter species has large patches of black pigment on this region. Fig. 8J Larval morphology. Overall body shape slender and slightly fusiform. Prostomium broad and rounded anteriorly. Three pairs of black eyes present; median eyes rounded, most lateral pairs double-eyes. Ramified melanophores between middle and lateral pair of eyes absent. Black pigment on lateral peristomium absent. Dorsal pigmentation consists of two rows of band-shaped melanophores from chaetiger II. These melanophores undergo expansion and contraction, expand to ramified melanophores or contract to non-ramified band-shaped melanophores. Lateral and ventral pigments absent. In late larvae modified chaetae develop in chaetiger V. Gastrotrochs on chaetigers III, V, VII, X, XIII, and XV.

Polydora cf. spongicola Berkley & Berkeley, 1950
Remarks. Adults of this species were collected from mud tubes constructed on the sponge Mycale sp. in Moroiso Bay, Misaki Peninsula (Table 1). The morphology of its modified spines in chaetiger V and the sponge-associated ecology of adults match the description of P. spongicola by Radashevsky (1993). However, this species was referred to P. cf. spongicola because the adult specimens were in poor condition, which hindered their morphology examination. The larvae and adults were confirmed to match (18S: 1770/1771, 16S: 474/475 bp) using molecular data (Fig. 2).
Only one planktonic larva of this species was collected in Sasuhama in January 2013. The larval morphology of P. cf. spongicola closely resembles that of P. spongicola described by Radashevsky (1988, as Polydora uschakovi Buzhinskaja, 1971) from Russia. Polydora uschakovi originally described from Russia was synonymized with P. spongicola (type locality: Canada) by Radashevsky (1993). Later, Blake (2017) described the larvae of P. spongicola from California and doubted this synonymization because, despite the similarities between the larvae from Russian and California, there are several morphological differences including the nature of the major spines of chaetiger V and the distribution of nototrochs and gastrotrichs. However, the larval dorsal pigment pattern of P. spongicola described by Blake (2017) greatly differs from those of P. cf. spongicola in the present study and of P. spongicola in Radashevsky (1988) but resembles that of P. brevipalpa in the present study. Conspecificity between P. uschakovi and P. spongicola should be verified in future studies.
Polydora sp. 1 Fig. 8K, L Larval morphology. Overall body shape slender. Prostomium broad and rounded anteriorly. Three pairs of black eyes present; median eyes rounded and lateral pairs double-eyes. Ramified melanophore between innermost and next to innermost pairs of eyes absent. Weak brown pigmentation on lateral parts of peristomium present or absent. Dorsal pigmentation consists of two rows of melanophores from chaetiger III, those of first five pairs band-shaped and remaining pairs dot-like in late larvae (Fig.  8L). These melanophores all dot-like in early larvae (Fig. 8K). Lateral pigment found on chaetigers II, IX, X, and XI in late larvae. Dorsolateral pigment at base of parapodia on posterior chaetigers. A pair of black and brown pigment patches occur on pygidium. Ventral brown pigment present on posterior part of late larvae. Pygidium has a dorsal notch and lacks appendages. Telotroch well developed. In late larvae, modified chaetae develop in 5 th chaetiger.
Remarks. Adults of this species are shell-borer and were collected from the shell of the turban snail O. rusticus in Sasuhama. The adults of this species have characteristic conspicuous black bars in their palps and are morphologically similar to Polydora neocaeca Williams & Radashevsky, 1999. Polydora haswelli previously recorded in Japan  was reexamined as P. neocaeca by comparing morphology and molecular sequences with the specimens from near the type locality (Malan et al. 2020). As the 18S and 16S rRNA gene sequences of Polydora sp. 1 and P. neocaeca showed differences (18S: 8/1771, 16S: 40/476 bp), the specimens collected in the present study were referred to a different species. Only two individuals of planktonic larvae of this species were collected in Onagawa Bay in April and July 2011. The larvae and adults were confirmed to 100% match using molecular data (Fig. 2). Fig. 8M Larval morphology. Overall body shape slender. Prostomium broad and rounded anteriorly. Three pairs of black eyes present, most lateral pairs double-eyes. Ramified melanophores between first and second innermost pairs of eyes absent. Pigmentation on lateral peristomium weak brown or absent. Dorsal pigmentation consists of two rows of melanophores from chaetiger II, with those of first 4-6 chaetigers being bandshaped and then replaced by ramified melanophores in posterior chaetigers. Dorsal pigments faint, undergo expansion and contraction, expand to cover almost whole of dorsal surface as finely ramified black pigmentation (Fig. 8M) or contract to bandshaped or dot-like black pigments without ramification. Faint lateral pigment found on late larvae on chaetigers VII onwards. Ventral pigments absent. A pair of brown pigments occur on the pygidium. Pygidium has a dorsal notch and lacks appendages. Gastrotrochs on chaetigers III, V, VII, X, XIII, XV, XVII, and XIX. Modified chaetae develop in chaetiger V in late larvae.

Polydora sp. 2
Remarks. No benthic adult stages were collected in the present study. These larvae formed a robustly supported monophyletic clade with other Polydora species (Figs 2, 3). Nevertheless, this species was identified as a member of Polydora. As the 18S and 16S rRNA gene sequences obtained from the larvae did not match any other available Polydora sequences, this species was referred to Polydora sp. 2.
Only two individuals of planktonic larvae of this species were collected from Sasuhama and Gamo Lagoon in January 2013. The faint dorsal pigmentation of the larvae of this species is unique among the members of Polydora collected in the present study. Fig. 8N-P Larval morphology. Overall body shape slender. Prostomium broad and rounded anteriorly. Three pairs of black eyes present; median pair of eyes rounded, most lateral pairs double-eyes, ramified melanophore between innermost and next to innermost pairs of eyes present. Large patches of black pigment located on lateral part of peristomium, behind prototroch. Dorsal pigment pattern consists of two rows of melanophores from chaetiger III with those of first four or five chaetigers being band-shaped and then replaced by ramified branching melanophores (Fig. 8O). These melanophores undergo expansion and contraction. Lateral pigment found on chaetigers II-IV, resumes again from chaetiger VII in late larvae (Fig. 8P). A pair of black pigment patches occur on pygidium. Ventral brown and black pigment present on posterior part in late larvae ready to metamorphose. Pygidium has a dorsal notch and lacks appendages. Telotroch well developed. Gastrotrochs on chaetigers III, V, VII, IX, X, XIII, XV, and XVII, lost on chaetigers III and V in late larvae (Fig. 8P). In late larvae, modified chaetae develop in chaetiger V.

Polydora sp. 3
Remarks. No benthic adult stages were collected in the present study. The 18S rRNA gene sequences obtained from the larvae did not match any available Polydora sequences. As the larvae formed a robustly supported monophyletic clade with other Polydora species (Figs 2, 3), this species was referred to Polydora sp. 3.
Planktonic larvae of this species were collected from December to June in Onagawa Bay every year during the study period. Planktonic larvae of this species were previously reported to be abundant in Onagawa Bay in the winter season from December to March (Abe et al. 2014, as Polydora sp.). Large patches of black pigment on the lateral peristomium are the main characteristic of this species and differentiate it from the other species of the genus observed in the present study, even at early planktonic stages (Fig. 8N).
Identification key to species of the larvae belonging to the genus Pseudopolydora in northeastern Japan  Radashevsky and Hsieh (2000) and Abe et al. (2016). However, the 16S rRNA gene sequence obtained in the present study showed a 11.5% (35/304 bp) difference with that of P. achaeta from Taiwan (country of type locality), which indicate that these two are different species. Therefore, this species is referred to P. aff. achaeta. The larvae and adults were confirmed to 100% match using molecular data (Fig. 2).
Planktonic larvae of this species with more than 3-chaetiger stages were abundant in Onagawa Bay during July to November (Abe et al. 2014). A dorsal pigmentation area shaped like "tilted wheels" is a unique characteristic of this species among the known Pseudopolydora larvae. Larval morphology. Overall body shape fusiform, head region enlarged due to broad prostomium and expanded lateral lips of vestibule. Prostomium gently notched anteriorly. Three pairs of black eyes present in more or less a straight line, most lateral pairs double-eyes. Mid-dorsal melanophore on first chaetiger usually absent (Fig. 9C), small non-ramified melanophore present in some individuals. Dorsal pigment consists of four rows of lateral and central pairs of pigment spots. Lateral and central pigments usually begin from chaetigers II and III, respectively. There pigment spots undergo expansion and contraction. Ventral pigment begins on chaetiger III, consists of paired bars on posterior border of each chaetiger. Anterior and posterior margin of prostomium have considerable brown pigment. Black pigment spots occur on sides of peristomium. Pygidium has black central spot. Gastrotrochs on chaetigers V and VII in 13-chaetiger larvae.
Remarks. Adult individuals of this species were collected from muddy sediment in Gamo Lagoon in January, May, and December 2011, and April 2013. Adult morphology agrees with the description of P. cf. kempi by Abe et al. (2016). Therefore, these individuals were referred to P. cf. kempi. The 16S rRNA gene sequence obtained in the present study showed a 99.7% (305/306 bp) similarity with that of P. kempi japonica Imajima & Hartman, 1964 from Russia (MG460897) reported by Radashevsky et al. (2020b), indicating these two are same species. It will need to be clarified whether P. kempi (type locality India) and subspecies P. kempi japonica (type locality Japan) are the same species. The larvae and adults were confirmed to 100% match using molecular data (Fig. 2).
Planktonic larvae of this species larger than 12-chaetiger stages were collected from Gamo Lagoon in August 2012. The larval morphology of this species observed in the present study agrees with the descriptions of P. kempi by Blake and Woodwick (1975) and of P. cf. kempi by Kondoh et al. (2017). These species have adelphophagic and lecithotrophic larval development, in which larvae feed on nurse eggs in brood capsules, hatch at a very late stage, and have a short pelagic life (Blake and Woodwick 1975;Kondoh et al. 2017). Reproduction and larval development of these species under the name of P. kempi and P. kempi japonica were also described by Srikrishnadhas and Ramamoorthi (1977), Myohara (1979), and Radashevsky (1985). However, the larvae of species in these descriptions resemble those of Pseudopolydora cf. reticulata Radashevsky & Hsieh, 2000 described by Kondoh et al. (2017) and of the present study in having planktotrophic development without nurse eggs and distinct dorsal melanophores including a middorsal melanophore on the first chaetiger. The taxonomy of P. kempi is unclear because its original description is quite brief, and the current location of type specimen is unknown (Radashevsky and Hsieh 2000). Therefore, studies resolving the taxonomy of P. kempi are necessary.
Pseudopolydora paucibranchiata (Okuda, 1937) Fig. 9D Larval morphology. Overall body shape fusiform, head region enlarged due to broad prostomium and expanded lateral lips of vestibule. Prostomium gently notched anteriorly. Three pairs of black eyes present in more or less a straight line, most lateral pair comma-shaped. A mid-dorsal ramified melanophore on chaetiger I. A pair of melanophores present dorso-laterally from chaetigers II onwards. Black pigment spots occur on lateral surface of chaetiger II, on sides of peristomium, and pygidium. Two small medial black pigment spots occasionally present ventrally on approximately chaetiger VI and anterior margin of pygidium. Gut has yellow-green color due to ingested food. Gastrotrochs on chaetiger V, VII, and XI in 13-chaetiger larvae.
Remarks. Adult individuals were collected from muddy bottom sediment in the intertidal zone of Mangoku-ura Inlet in July 2014. Adult morphology agrees with the description of Pseudopolydora paucibranchiata by Okuda (1937, as Polydora). Therefore, these individuals were referred to this species. The larvae and adults were confirmed matching (18S: 1784/1784, 16S: 454/455 bp) using molecular data (Fig. 2).
The planktonic larvae of this species were reported to be common in Onagawa Bay during June to November (Abe et al. 2014). The larval morphology of this species observed in the present study agrees with the descriptions by Blake and Woodwick (1975), Ward (1977), Myohara (1980), Wu and Chen (1980), Radashevsky (1983), andBlake (2006). The dorsal pigment pattern of this species consists of one pair of melanophores, which agrees with that of the larvae of Pseudopolydora vexillosa Radashevsky & Hsieh, 2000photographed by Mok et al. (2009) and Chandramouli et al. (2011, 2013, currently synonymized to P. paucibranchiata (Junqueira et al. 2009). The dorsal pigment pattern of these larvae is also similar to that of the larvae of Pseudopolydora antennata (Claparède, 1869) described by Hannerz (1956, as Polydora antennata), but the latter species has a more thickened body shape compared to the former. Fig. 9E, F Larval morphology. Overall larval shape fusiform, head region enlarged due to broad prostomium and expanded lateral lips of vestibule. Prostomium slightly notched anteriorly. Three pairs of black eyes present in more or less a straight line, most lateral pairs double-eyes. Large patches of black pigment on lateral peristomium present. Middorsal melanophore on chaetiger I usually present. Dorsal pigments undergo expansion and contraction, expanding to cover almost complete dorsal surface with finely ramified black pigment (Fig. 9F) or contract to dot-like black pigmentation without ramifications (Fig. 9E). Ventral pigment usually absent, consisting of paired bars on the posterior border on anterior chaetigers occasionally present. Black pigment on pygidium. Gastrotrochs on chaetigers V, VII, and XII in 17-and 18-chaetiger larvae, late larvae lose gastrotrochs on chaetigers V and/or XXII.

Pseudopolydora cf. reticulata Radashevsky & Hsieh, 2000
Remarks. Adult individuals of this species were collected from muddy sediment in Gamo Lagoon in April 2013 and Sasuhama in July and September in 2011. Adult morphology agrees with the description of P. cf. reticulata by Abe et al. (2016). Therefore, these individuals were referred to this species. The 16S rRNA gene sequence obtained in the present study showed a 99.4% (304/306 bp) similarity with that of P. bassarginensis (Zachs, 1933) from Russia (MG460894) reported by Radashevsky et al. (2020b), indicating these two are one species. Although the Japanese population shows intermediate morphological characteristics between P. reticulata (type locality Taiwan) and P. bassarginensis (type locality Russia), Abe et al. (2016) tentatively identified the Japanese population as P. cf. reticulata because the original description of P. bassarginensis is very brief and the status of the species remains unclear. The results of the present study indicate that the Japanese population likely belongs to P. bassarginensis, but whether the morphologically similar P. reticulata and P. bassarginensis are considered molecularly as the same or different species will need to be clarified. Planktonic larvae of P. cf. reticulata larger than the 3-chaetiger stage were collected from Gamo Lagoon, Sasuhama, and Onagawa Bay mainly from July to September. The larvae and adults were confirmed to match (18S: 1775/1775, 16S: 468/470 bp) using molecular data (Fig. 2).
Pseudopolydora cf. reticulata and P. cf. kempi are very similar sister species; specimens from Japan once misidentified as P. cf. kempi or P. kempi japonica were distinguished based on their morphology and 18S and 28S rRNA gene sequences by Abe et al. (2016). The larvae of these two species are also quite similar, but the mid-dorsal melanophore on chaetiger I is usually present in Ps. cf. reticulata and absent in P. cf. kempi; moreover, the dorsal pigmentation is more distinct in the former species than in the latter. The two species also differ in reproduction and larval development: P. cf. kempi has lecithotrophic development with a short planktonic phase, whereas P. cf. reticulata has planktotrophic development with a long planktonic phase (Kondoh et al. 2017). Fig. 9G, H Larval morphology. Overall larval shape fusiform, head region enlarged due to broad prostomium and expanded lateral lips of vestibule. Prostomium gently notched anteriorly. Three pairs of black eyes present, comprising one pair of rounded median eyes, one pair of large lateral eyes, and one pair of large anterior eyes. Lateral and anterior pairs of eyes link with each other and form dumbbell-shapes almost divided into two equal parts by a deep constriction. Mid-dorsal ramified melanophore present on chaetiger I in early larvae. Mid-dorsal melanophore on chaetiger I occasionally absent or expanded to finely ramified melanophore in late larvae. A paired of melanophores occur dorso-laterally from chaetiger II onwards, usually finely ramified in late larvae (Fig. 9H). Ramified melanophores cover almost entire ventral surface on chaetigers III-VII in 11-chaetiger larvae. Black pigment spots on sides of peristomium absent. Conspicuous large black pigment on pygidium. Gastrotrochs on chaetigers V and VII in 11-chaetiger larvae.

Pseudopolydora tsubaki Simon, Sato-Okoshi & Abe, 2017
Remarks. Adult individuals were collected from mud deposits in crevices of shells of living C. gigas oysters in Habu Port, Izu-Oshima Island, and Tomiura, Boso Peninsula in April 2016. Adult morphology agrees with the description of Pseudopolydora tsubaki by Simon et al. (2019a). Therefore, these individuals were identified as P. tsubaki. The larvae and adults were confirmed to 100% match using molecular data (Fig. 2).
A small number of planktonic larvae of this species were collected in Habu Port and Tomiura in May and June 2016. The larvae of P. tsubaki are similar to those of Pseudopolydora pulchra (Carazzi, 1893) in having ramified melanophores covering the ventral side; however, these cover only the central part of the body in the former species, whereas those of latter species cover the ventral surface almost entirely (Hannerz 1956, as Polydora pulchra;Rullier 1963, as Polydora pulchra). The dorsal pigment pattern is also different in these two species: two pairs of melanophores are distinct in P. pulchra, whereas the melanophore pair is ambiguous in P. tsubaki. Pseudopolydora sp. Fig. 9I Larval morphology. Overall larval shape slightly fusiform, head region enlarged due to broad prostomium and expanded lateral lips of vestibule. Prostomium rounded anteriorly. Three pairs of black eyes present, comprising one pair of rounded median eyes, one pair of large lateral eyes, and one pair of large anterior eyes. Lateral and anterior pairs of eyes link with each other and form a dumbbell-shape almost divided into two equal parts by a deep constriction. Small mid-dorsal melanophore present on chaetiger I. A distinct paired melanophore occurs dorso-laterally from chaetiger II onwards, ramified in anterior chaetigers. Black pigment spots on sides of peristomium absent. Dot-like black pigmentation on pygidium.
Remarks. No benthic adult stages were collected in the present study. The larvae formed a monophyletic clade with the other Pseudopolydora species with > 50% bootstrap support (Figs 2, 3). Therefore, this species was identified as a member of Pseudopol-ydora. As the 18S rRNA gene sequences obtained from the larvae did not match any of the available Pseudopolydora sequences, this species is referred to Pseudopolydora sp.
Only one larva individual was collected from Sasuhama in August 2011. The dorsal pigment pattern of this larva somewhat resembles that of P. paucibranchiata; however, the mid-dorsal pigment of this species is weaker and its dorsolateral melanophores are more ramified than those of P. paucibranchiata. The eye arrangement of this larva resembles that of late P. tsubaki larvae: three pairs of black eyes are present, but not in a straight line.

Genus Spio Fabricius, 1785
Larval diagnosis. Overall body shape long, slender, and weakly or moderately fusiform. Prostomium small and rounded anteriorly. Lateral part of peristomium weakly demarcated from prostomium. Three pairs of black eyes present, most lateral often double-eyes. Dorsal pigmentation consists of transverse band-shaped or dot-like paired lateral melanophores. Some species lack black pigmentation. Ventral pigment usually absent. Dark-brown pigment may be present on pygidium. Nototrochs occur in all chaetigers except first one or two chaetigers, where nuchal organs develop. Gastrotrochs occur regularly in every other chaetiger from chaetiger III onwards. Larval chaetae on first chaetiger usually fairly long. Branchiae develop in late larvae, first on chaetiger II or III. One pair of anal cirri present on pygidium in late larvae (Thorson 1946, as spionid larva C, E, and F;Hannerz 1956;Wu et al. 1965;Simon 1963Simon , 1967Simon , 1968Guérin 1972;Srikrishnadhas and Ramamoorthi 1981;Plate and Husemann 1994).
Identification key to species of the larvae belonging to the genus Spio in northeastern Japan Remarks. Adult individuals of this species were collected from Rishiri Island, northern Japan, in July and August 2017. These specimens were previously identified as S. arndti Meißner, Bick & Bastrop, 2011(Abe et al. 2019c) since adult morphology agreed. Although 18S rRNA gene sequence obtained in the present study 100% match with that of S. arndti (FR823434, 1761/1761 bp), because the 16S rRNA gene sequences were different (6.7%, 30/451 bp), the species reported here is referred to Spio sp. 1. The 16S rRNA gene sequence of Spio sp. 1 was rather more similar (96.1%, 298/310 bp) to that of Spio sp. 2573 from Russia (KT200126), but conspecificity of these two is unclear. A few planktonic larvae of this species were collected from Onagawa Bay only in May 2011. The larvae and adults were confirmed to match (18S: 1762/1762, 16S: 466/467 bp) using molecular data (Fig. 2).
The absence of distinct black melanophores in larvae of this species differentiates them from those of Spio sp. 2 (see below). Slight dorsal pigmentation was also reported in adelphophagic benthic larvae of Spio setosa Verrill, 1873 sensu Simon (1967Simon ( , 1968, which were essentially unpigmented, and in those of Spio multioculata (Rioja, 1918) described by Hannerz (1956). However, the larval morphologies of these two species are different from that of Spio sp. 1 in lacking ventral black pigment on the peristomium (in both former species) and long larval chaetae on the first chaetiger (in S. setosa), and in having a relatively thickened body shape (in both species). Fig. 9L-N Larval morphology. Overall larval shape elongated, slender, weakly fusiform. Larval chaetae on first chaetiger fairly long. Prostomium round anteriorly. Small patches of black pigment on lateral peristomium present ventrally. Three pairs of black eyes present, most lateral pairs double-eyes. Two rows of dot-like black melanophores on each side of dorsum from chaetiger I onwards, linking by band-shaped medial black pigmentation from chaetiger IV or V. Pharynx exhibits weak dark or brownish pigmentation. The larvae which are ready to metamorphose have branchiae from chaetiger II, pigment spot on palps, and a pygidium with four leaf-shaped anal cirri.

Spio sp. 2
Remarks. Adult individuals were collected from muddy sand sediments of shallow water in Sasuhama in September 2011. These adults were morphologically identified as a Spio species, but they could not be identified to species level. Spio spp. 1 and 2 are distinguishable morphologically by the number of ventral epidermal glands. The 18S and 16S rRNA gene sequences obtained in the present study did not match any of the available Spio sequences (Figs 2, 3). The larvae and adults were confirmed to 100% match using molecular data (Fig. 2).
Planktonic larvae of this species were found in Sasuhama and Onagawa Bay from April to August during the study period. Larval morphology and pigmentation pattern of this species is similar to that of Spio decorata Bobretzky, 1870 described by Guérin 1972. However, the latter species was originally described from the Black Sea and has not been recorded in Japan.

Larval identification based on the molecular data
The present study identified 41 species from 14 genera of planktonic spionid larvae by comparing adult and larval gene sequences and revealed high diversity of spionid larvae in neritic plankton communities (Table 1, Figs 2, 3). Planktonic spionid larvae of several species could not be identified to species level because of the lack of adult reference sequences or difficulties in adult identification. As the genetic information available for many marine invertebrate taxa including polychaetes is insufficient, the increase in gene sequence data based on accurate species identification and the establishment of a comprehensive database of adult reference sequences are essential for a more precise and efficient larval molecular identification. However, most of the larvae from the present study that did not have sequences that matched those of adults were identified to genus level based on their position within the phylogenetic tree; this was only possible because many of the spionid genera were recovered well or moderately supported monophyletic groups in our molecular phylogenetic analyses (Figs 2, 3). In contrast, the monophyly of some spionid taxa, particularly of the genera Dipolydora, Malacoceros, and Prionospio were ambiguous and not well supported in the phylogenetic tree recovered herein. It should be noted that Malacoceros indicus and Malacoceros cf. indicus were recovered as quite distant from Malacoceros fuliginosus and Malacoceros sp. (Fig. 3), potentially indicating the paraphyletic origins of these two clades. The results of the phylogenetic analyses also showed that the monophyly of subfamily Nerininae is doubtful and more likely to be paraphyletic. Because intergeneric phylogenetic relationships were ambiguous due to the low statistical support of most of the higher internal nodes (Figs 2, 3), it was difficult to compare with the previous results of phylogenetic relationships among spionid genera provided by Sigvaldadóttir et al. (1997) and Blake and Arnofsky (1999). The results of our phylogenetic analyses reinforce the need for a more robust and comprehensive molecular phylogenetic study of this taxon to test the monophyly of each genus and subfamily and to shed light on the phylogenetic relationships among spionid genera.
In the present study, many spionid species were collected as planktonic larval stages. This emphasizes the effectiveness of field investigations of both larval and adult stages to assess the cryptic species diversity in benthic invertebrate fauna of coastal waters. The reference gene sequences used in the present study for adults covered most of the species belonging to the genera Polydora and Pseudopolydora hitherto recorded from Japan (Sato-Okoshi 1999Abe 2012, 2013;Teramoto et al. 2013;Abe et al. 2016;Simon et al. 2019a). However, the sequences of some Polydora and Pseudopolydora larvae, namely Polydora sp. 2, Polydora sp. 3, and Pseudopolydora sp., did not match any adult reference sequences. This emphasizes the need for detailed taxonomic studies with a more comprehensive sampling of spionid adults to reveal the actual biological diversity of this taxon in Japan.

Morphology of spionid larvae
The family Spionidae can be divided into two subfamilies: 1) Spioninae Söderström, 1920, which includes the genera Spio, Microspio Mesnil, 1896, Pygospio Claparède, 1863, and genera of the tribe Polydorini; and 2) Nerininae Söderström, 1920, which includes almost all remaining spionid genera, except for Atherospio Mackie & Duff, 1986and Pygospiopsis Blake, 1983(Blake 2006, besides Poecilochaetus and Trochochaeta, which were recently placed within the family Spionidae (Radashevsky et al. 2018). The larvae of these two subfamilies were distinguished in the present study based on color and number of eyes, body pigmentation, shape of peristomium, and distribution of gastrotrochs, and by the following characteristics identified by Hannerz (1956): larvae of Spioninae have three pairs of black eyes (lateral eyes are often double eyes), distinct black pigmentation with melanophores, lateral parts of the peristomium not demarcated from prostomium, and gastrotrochs present from chaetiger III, V, or VII onwards, but absent in all of the succeeding chaetigers (Figs 7-9); larvae of Nerininae have two pairs of red or dark red eyes, lack distinct black pigmentation, lateral parts of the peristomium are well developed and often demarcated from prostomium, and gastrotrochs present from chaetiger II or III onwards and in all succeeding chaetigers (Figs 4,5). Blake (1969) also discussed the presence of ventral ciliary patches in early larval stages as a common characteristic of subfamily Spioninae, but these cilia were not herein observed because they are lost in early larval stages. Hannerz (1956) reported the following exceptions to the abovementioned typical larval morphologies: larvae of Prionospio fallax Söderström, 1920 (as P. malmgreni, see Blake andArnofsky 1999) with two pairs of black eyes; larvae of Malacoceros (as Scolelepis), which belongs to Nerininae, with intermediate characteristics between the two subfamilies, i.e., with three pairs of black eyes and gastrotrochs regularly distributed on every other chaetiger as in Spioninae larvae. However, Plate and Husemann (1994) reported that the larvae of Malacoceros fuliginosus (Claparède, 1868) have up to three pairs of red eyes in early stages and that eye color changes to black as larvae develop. Radashevsky and Migotto (2006) reported that the larvae of Malacoceros sp. have two pairs of red eyes. In the present study, the larvae of Rhynchospio have two pairs of dark red eyes (Fig. 4F, G), whereas their morphology resembled those of Malacoceros species described by Hannerz (1956). Radashevsky (2007) also reported that larvae of Rhynchospio nhatrangi have two pairs of red eyes. Besides the various reports on the number and color of eyes, the close relationships of Malacoceros and Rhynchospio to the subfamily Spioninae were indicated by larval morphology, and results of the phylogenetic analyses presented (Figs 2, 3) also provide some support for this hypothesis.
In the subfamily Spioninae, the most obvious larval differences between genera and species are the overall body shape and type and arrangement of pigmentation (Blake and Arnofsky 1999). The overall body shape of larvae of Polydora, Dipolydora, and Spio tended to be long and slender, whereas those of Boccardiella, Boccardia, and Pseudopolydora tended to be thick and fusiform (Figs 7-9), although Boccardia proboscidea (Fig. 7A, B) and Boccardia sp. 1 (Fig. 7D-F) showed relatively slender body shapes. The lateral enlargement of the prostomium in Spioninae is variable: large in Boccardiella and Pseudopolydora, moderate in Polydora and Boccardia, and small in Dipolydora and Spio. Fairly long larval chaetae on the first chaetiger are highly characteristic of Spio within Spioninae.
The dorsal black pigmentation with melanophores is distinct in the subfamily Spioninae, and the pattern of rows of melanophores is generally diagnostic among Spioninae genera. The typical patterns of dorsal pigmentation rows in larvae are as follows: a pair of transverse bands of black pigment on some anterior chaetigers followed by a pair of large branching melanophores in Polydora; lack of large melanophores, but with a pair of medial spots or bands, a pair of lateral pigment patches, and mid-dorsal black pigment spot continuing posteriorly from the anterior chaetigers in Dipolydora; mid-dorsal melanophores arranged in a single row in Boccardia; medial and lateral pairs of spots or bands with black pigmentation and a small patch of pigment at the base of the notopodia present on almost all chaetigers in Boccardiella; a mid-dorsal melanophore on the first chaetiger, and one or two pairs of melanophores on each chaetiger in Pseudopolydora; a pair of black pigment spots and transverse black pigment bands linking them on each chaetiger in Spio (Figs 7-9). These typical dorsal pigment patterns were also reported in many previous studies (Hannerz 1956;Blake 1969Blake , 2006Blake and Arnofsky 1999; and references cited therein). However, unusual larval pigment patterns are often found in members of each of the aforementioned genera; therefore, these typical larval pigment patterns are not wholly consistent within each genus. For example, the single row of dorsal melanophores typical of Boccardia larvae was also observed in larvae of Polydora cf. glycymerica (Fig.  8E) and Dipolydora cf. commensalis (Fig. 7L), and have been reported in Polydora glycymerica (Radashevsky 1989), Polydora hermaphroditica (Hannerz 1956;Plate and Husemann 1994), Dipolydora commensalis (as Polydora commensalis : Andrews 1891;Hatfield 1965;Blake 1969;Radashevsky 1989), and Pseudopolydora rosebelae Radashevsky & Migotto, 2009. In contrast, the single row of dorsal melanophores is absent in Boccardia chilensis Blake & Woodwick, 1971(Carrasco 1976Blake and Kudenov 1981), Boccardia pseudonatrix (Fig. 7C), and Boccardia semibranchiata Guérin, 1990(Guérin 1991. The larvae of Pseudopolydora cf. kempi lack a mid-dorsal melanophore on the first chaetiger, which is typical in Pseudopolydora larvae ( Fig. 9C; Kondoh et al. 2017). The distinct dorsal black pigment is absent in Spio setosa Verrill, 1873 (Simon 1967(Simon , 1968 and Spio sp.1 (Fig. 9J, K).
Larvae of the following Spioninae genera were not collected in the present study: Microspio, Pygospio, and the polydorid genera Amphipolydora Blake, 1983, Carazziella Blake & Kudenov, 1978, Polydorella Augener, 1914, and Tripolydora Woodwick, 1964 (among them, Microspio and Carazziella have records from Japan by Okuda 1937, Sato-Okoshi 1998. Little is known about the larval morphology of the genera Amphipolydora, Polydorella, and Tripolydora. The larvae of Microspio resemble those of Spio in having a long and slender body shape and band-shaped dorsal black pigmentation (e.g., Hannerz 1956;Cazaux 1971). The larvae of Carazziella resemble those of the polydorid genus Boccardia in having a fusiform body shape and a single row of dorsal melanophores (Carrasco 1976, as Polydora citrona;Blake and Arnofsky 1999;Blake 2006). The morphology of planktonic larval stages of Pygospio elegans as described in Hannerz (1956) resembles that of Pseudopolydora in having a thick and fusiform body shape, laterally enlarged prostomium, and mid-dorsal melanophore on the first chaetiger. Blake (1969) also noted the morphological similarity between the larvae of Pseudopolydora and Pygospio elegans and suggested the possibility that polydorids are closely related to Pygospio through Pseudopolydora. Subsequently, Blake and Woodwick (1975) reported the similarities of nurse egg feeding patterns between Pseudopolydora kempi and Pygospio elegans, further strengthening the view of a close relationship between these two genera. This hypothesis is supported by the results of the phylogenetic analysis presented, showing that polydorids plus Pygospio form a monophyletic clade with robust statistical supports (Fig. 3).
In the subfamily Nerininae, as in Spioninae, the most obvious differences among genera are also regarding their overall body shapes. The lateral parts of the peristomium are conspicuous, well developed, and distinctly demarcated from the prostomium in larvae of Laonice, Rhynchospio, and Scolelepis, but they are less pronounced in those of Aonides, Paraprionospio, Prionospio, and Spiophanes, as previously noted by Hannerz (1956). Larvae of the former group of genera (Laonice, Rhynchospio, Scolelepis) also have a relatively wide body shape, whereas those of the latter group have a narrow body shape. Regarding the larvae of the former group, the prostomium is more or less stumpy and not pointed anteriorly in Rhynchospio and Laonice; however, Scolelepis larvae have a unique body shape distinct from other spionid genera and their prostomium is pointed anteriorly, terminating in a tapered tip, and the lateral parts of the peristomium are demarcated and bearing a large peristomial umbrella. The larvae from the latter group (Aonides, Paraprionospio, Prionospio, Spiophanes), Paraprionospio, and Prionospio characteristically have extremely long and thin bodies with numerous chaetigers. In particular, larvae of Paraprionospio are extremely large in terms of body size and chaetiger number at metamorphosis among the spionid larvae (Yokoyama 1981). The larval morphology of the genus Poecilochaetus resembles that of Paraprionospio and Prionospio: larvae have extremely long and slender transparent bodies without distinct black pigmentation. However, the first differs from the other two in having small lateral pigment spots on each side of the chaetigers, a long metatrochophore stage (up to ca. 30-40 chaetiger stages), and a serpentine swimming behavior with developed parapodia bearing cirriform dorsal and ventral postchaetal lobes in the nectosoma stage. Poecilochaetus larvae are distinctive among spionid larvae in having gastrotrochs from chaetiger I onwards despite all other Nerininae larvae having gastrotrochs from chaetiger II or III onwards. In the present study, a pair of lateral processes on the prostomium developed in late larvae was found only in larvae of Rhynchospio and Spiophanes, it also previously described for Malacoceros larvae (Hannerz 1956). Fairly long and straight larval chaetae on the first chaetiger are highly characteristic of Aonides larvae, especially in the early stages (Fig. 4A, B); however, similar long and straight larval chaetae were also herein observed in larvae of Spio sp. 2 (Fig. 9L, M). Although larvae of the genera Malacoceros, Marenzelleria Mesnil, 1896, Streblospio Webster, 1879, and Trochochaeta (subfamily Nerininae) were not collected in the present study, larval morphologies of these genera have been well described in previous studies. The larvae of Malacoceros resemble those of Rhynchospio (see above). The larvae of Marenzelleria (Bochert and Bick 1995) resemble those of Laonice in having the following characters: the remains of the egg membrane visible in early stages; in late larvae, lateral parts of the peristomium are conspicuous, well developed and distinctly demarcated from prostomium, and palps start developing laterally on the peristomium; the body is broader than the prototroch; both notopodial and neuropodial larval chaetae are present. However, the larvae of these two genera differ in the arrangement of nototrochs and gastrotrochs. The larval morphology of the genus Streblospio, which is included in the Prionospio complex (Dean 1965;Blake and Arnofsky 1999;Blake 2006) resembles that of Prionospio. The larval morphology of the genus Trochochaeta is distinctive among spionid larvae in having unusually long larval chaetae on the first chaetiger, very pronounced peristomial umbrella with two rows of robustly developed prototrochs, and total absence of nototrochs; larvae of this genus also present the typical morphological characteristics of Nerininae larvae, such as two pairs of red eyes, lack of distinct black pigmentation, and gastrotrochs from chaetiger II onwards on all succeeding chaetigers, although gastrotrochs of Trochochaeta larvae are weakly developed and those on chaetiger II are especially small and inconspicuous in late-stage larvae (Hannerz 1956, as Disoma;Blake and Arnofsky 1999;Blake 2006).
There is insufficient information on the larval morphology of the remaining genera of Nerininae. The larval development and morphology of Dispio uncinata Hartman, 1951 were described, and this species' close relationship with Aonides was suggested by Blake and Arnofsky (1999) and Blake (2006). However, Radashevsky et al. (2011) pointed out that the larvae described by these authors are most likely those of Aonides californiensis Rioja, 1947 rather than of a Dispio Hartman, 1951 species. Species-level identification of larvae from this subfamily is generally more difficult because of the lack of structured pigmentation, which is a useful characteristic for identifying species of Spioninae larvae. Especially in the genus Prionospio, larval morphology is quite simple and similar among species, which made it impossible to find morphological characters to distinguish between them in the present study.
Except for Prionospio spp., most of the planktonic spionid larvae collected in the present study have morphological characteristics that could be used to distinguish genera and species, and allowed morphological identification based on overall body shape and pigment patterns. The present paper provides identification keys to genera and species of planktonic spionid larvae from northeastern Japan; however, sufficient attention to developmental and/or intraspecific variation of larval morphological characteristics and the disappearance of pigments after fixation (only the black pigment usually remains after fixation) is required for accurate larval identification.