Checklist of the micromolluscs in the intertidal zone of the Yellow Sea and Bohai Sea, China

Abstract Background The diversity of the sedimentary environment and molluscs is rich in the intertidal zone of the Yellow Sea and Bohai Sea. At present, many of the investigations focus on macromolluscs, while the diversity of micromolluscs is seriously underestimated. New information In this study, the survey of micromolluscs was carried out in the intertidal zone of the Yellow Sea and Bohai Sea. The collection and preservation of micromolluscs, as well as the preparation methods of morphological characteristic structures by scanning electron microscopy (SEM) were explored. A total of 20 species were described in this survey. These can be assigned to 16 families, four orders (Vetigastropoda (1), Caenogastropoda (10), Heterobranchia (5) and Autobranchia (3)) and two classes (Gastropoda (17), Bivalvia (3)).


Introduction
Molluscs are one of the largest groups of marine benthos, with a huge variety of life strategies.In marine ecosystems, molluscs occupy different trophic levels, from filtrators and phytophages to predators and parasites.Due to their high diversity and widespread distribution, they have been widely recorded in taxonomic monographs.Recent examples of taxonomic monographs from the Western Pacific Region included Liu (2008), Zhang et al. (2016), Okutani (2017), andLi et al. (2019).These monographs recorded a large number of taxa, but most of them are macromolluscs, while micromolluscs are not fully understood, especially in China.
Micromolluscs are important components of molluscs and play an important role in biodiversity assessments.As micromolluscs usually require specific collecting/sorting attention and have a reputation for presenting formidable taxonomic difficulties, they are often overlooked in biodiversity surveys, resulting in a gross underestimation of mollusc diversity.In a biodiversity survey of New Caledonia, the micromolluscs account for approximately one-third (33.5%) of all identified mollusk species, with the largest size class being 1.9-4.1 mm (Bouchet et al. 2002).Therefore, it is of great significance to carry out micromolluscs' surveys to assess the diversity of molluscs.
The diversity of the sediment environment and molluscs is rich in the intertidal zone of China.However, much of the literature focuses on macromolluscs that can be collected by hand-picking in the field.Only Liu (2013) investigated the small gastropods in the Zostera marina in Swan Lake, Rongcheng Bay China.Here, we carried out the survey of micromolluscs in the intertidal zone along the coasts of the Yellow Sea and Bohai Sea, established methods for sample collection and preservation, used morphological and molecular data to identify the species and performed morphological descriptions based on SEM.

Taxon sampling and processing
Sampling was carried out in the intertidal zone of the Yellow Sea and Bohai Sea from September 2017 to December 2019 (Fig. 1, Table 1).Sample collection was performed with reference to the handbook by Li et al. (2019).Field collection of micromolluscs requires some specialised techniques depending on the likely habitats of the target organisms and a range of microhabitats.Shell grit or shell sand The sediments usually contain mainly empty shells and certain species should be divided into size fractions by using graded sieves (e.g. 10, 5, 2.5, 1.0 and 0.4 mm mesh size).If there is a need to collect all adult species, the 0.4 mm sieve is suitable.

Algal samples
Algae are one of the most important habitats for micromolluscs.After the algae are collected on-site, it should be placed in a bucket or 'zip-lock' bag and then the bucket or bag should be shaken violently.The algal material is then removed and the sample is allowed to settle briefly.The water can then be gently decanted, being run through a sieve to catch any floating molluscs.

Rock
The upper and undersides of rocks are very different environments.Some micromolluscs may be attached to the algal films or turf on the rock surface.These rocks can be scrubbed with a brush in a bucket.Then the residue in the bucket is collected into the sample bottle.

Figure 1.
Map of sampling site.For details on collection sites, see Table 1.
Specimens for anatomical study were stored in formalin, whereas for molecular work, strong (> 95%) ethanol or RNALater are the best preservatives.All samples were brought back to the laboratory for further processing.All the material collected has been deposited in the Laboratory of Shellfish Genetics and Breeding (LSGB), Fisheries College, Ocean University of China, Qingdao, China.Morphological identification of the species was carried out mainly through the identification book from Okutani (2017) and original literature, as well as WoRMS (http://www.marinespecies.org).

Molecular analyses
Traditional barcoding gene COI was analysed for most specimens to verify identification.Total genomic DNA was extracted from entire animals with the TIANamp Marine Animals DNA Kit (Tiangen Biotech, Beijing, China), according to the manufacturer's protocol and stored at -4°C for short-term use.Amplification of partial sequences of mitochondrial COI was amplified by a polymerase chain reaction using the primers from Folmer et al. (1994), LCO1490 (5'-GGTCAACAAATCATAAAGATATTGG-3') and HC02198 (5'-TAAACTT CAGGGTGACCAAAAAATCA-3'). PCR reactions were made in a volume of 25 μl, using the following: 19.3 μl distilled H O (sterile MilliQ), 0.5 μl dNTPs (2.5 mM), 2.5 μl 10×buffer(Mg plus), 0.2 μl TakaRa Taq DNA polymerase, 1 μl of each primer (10 μM) and 0.5 μl genomic DNA (50-100 ng).Thermal cycling conditions were: 94℃ for 3 min, followed by 37 cycles of 94℃ for 45 s, 48℃ for 45 s, 72℃ for 60 s and a final extension of 72℃ for 10 min.PCR products were run in a 1.5% agarose gel to corroborate the success of the amplifications and sequenced by Shanghai Bioengineering Co., Ltd (China).DNA sequences were assembled using SeqMan (Swindell and Plasterer 1997).The obtained sequences were compared to sequences of molluscs in both GenBank and BOLD databases.Sequences have been deposited in GenBank (Table 2).

Preparation of micromollusc shells for SEM
Specimens can be immersed for one to two minutes in strong commercial bleach to remove the periostracum.The soaked shells were placed in a centrifugal tube with neutral detergent and cleaned by ultrasound.The cleaning time depends on the amount of dirt on the shell surface.After sonication, shells should be washed in distilled water preferably two or three times.After the last wash, the water drops should be removed with a paper towel and the specimen air-dried.The clean shells were attached to conductive glue in the direction of the standardized views and then sputtered with gold.The thickness of the metal coating is 1-10 nm; however, even excessive coating will not interfere with the detail in a normal shell.The operating tool was TESCAN VEGA3 scanning electron microscope (SEM).

Checklist of the micromolluscs in the Yellow Sea and Bohai Sea, China
Lirularia iridescens Schrenck, 1863 Diagnosis: Shell minute (2.5±0.26mm in width, 2.2±0.15mm in length), ovato-conica.Whorls 4-5, spire low, body whorl rather ventricose (Fig. 2).Suture distinct.Every whorl is striated by about 9-10 distinct spiral ribs and the distance between the spiral ribs is not uniform.Reddish-brown spiral ribs contrasting with iridescent interspaces; lines on ribs occasionally broken by white spots, with crater-like pitted microsculpture on protoconch.Aperture ovate, outer lip thin, with small notches, inner lip simple and slightly convex.Umbilicus circular deep.Basal surface flat, with 9-10 circular spiral ribs.
Native status: Found on seagrass between tidal and shallow waters.
Distribution: Yellow Sea of China; Japan Sea, South Kurile Islands.
Notes: L. iridescens was recorded by Kantor and Sysoev (2006).Okutani (2017) described and illustrated in detail L. iridescens.In this study, the species was redescribed, based on the SEM.Crater-like pitted microsculptures on the protoconch were first discovered here.At present, there are very few morphological studies on this genus Lirularia.Many studies have shown that L. iridescens are phytal gastropods inhabiting seaweeds (Toyohara et al. 1999, Agatsuma et al. 2005).Lirularia iridescens Schrenck, 1863.
Native status: Found on seagrass between tidal and shallow waters.Checklist of the micromolluscs in the intertidal zone of the Yellow Sea ...

Native status:
Lives on intertidal sandy mud, algae.
Notes: Stenotis lois (Yen, 1936) is very similar to Lacuna carinifera (A.Adams, 1853), but the aperture is wider, with angulate periphery on the underside of the outer lip, the umbilicus more open.This study thinks Stenotis lois (Yen, 1936) is the larva of Lacuna carinifera (A.Adams, 1853) and is a synonym.
Native status: Lives on algae in intertidal and sublittoral zones.

Barleeia sp.
Diagnosis: Shell minute (1.2±0.42 mm in width, 1.8±0.13mm in length), conic to ovateconic, dark brown, smooth or with weak axial microsculptures and rather solid, periphery weak convex to angled (Fig. 6).Whorls 4, suture prominent and deep.Protoconch dome-shaped, smooth, with a diameter of about 300 μm, some sediment attached to it, with several pits on the surface.Apical angle around 45°. Aperture oval with a simple peristome, the posterior of outer lip in contact with the body whorls.Operculum, not a canal; inner lip attaching to the previous whorl, but not contacted with outer lip, not umbilicate and anterior canal; outer lip thin, anterior prosocline.
Native status: Lives on algae in intertidal and sublittoral zones.
Checklist of the micromolluscs in the intertidal zone of the Yellow Sea ...

Iravadia elegantula A. Adams, 1861
Diagnosis: Shell minute (3.0±0.22 mm in length, 1.6±0.14mm in width), elongate conical, stout (Fig. 7).Whorls 5, inflated, with deeply impressed suture.Whorls increasing gradually in size.Surface of shell glossy dark brown at apical and yellowishbrown at abapical part, sculptured with many fine spiral lirae and fine growth lines.Periphery of body whorl rounded, base of whorl gradually curved.Protoconch is small, depressed dome-shape.Aperture oval and weakly angled both up and down, peristome somewhat thickened and broad down region, outer lip rounded.Umbilicus narrow and very shallow.
Native status: Brackish water, on muddy flats and under rocks in estuary at river mouth.

Stenothyra glabra A. Adams, 1861
Diagnosis: Shell minute (2.1±0.14 mm in length, 1.5±0.07mm in width), ovate-conic, rather thick, dorso-ventrically compressed, with rounded to angled inflation of last whorl (Fig. 8).Up to 5 whorls including protoconch, with less convex whorls and sutures moderately deep.Surface smooth, yellowish-brown, sculpture not dotted lines, but continuous spiral grooves.The aperture abruptly descending, contracted and near circular; peristome continuous, showing a weak triangular area above; outer lip round with marked grooves.Operculum ovate, yellowish, translucent, with very weak angulation aligning with posterior apex of aperture; exterior surface with central paucispiral nucleus close to the inner lip.Protoconch dome-shaped, smooth, 1-3/4-2 whorls; some pits in the first whorl.
Native status: Inhabiting the surface of mud flats or attaching to the under surface of floating leaves in freshwater estuaries.
Distribution: Yellow Sea and Bohai Sea of China, Korea and Japan.

Notes:
The type locality of Stenothyra glabra A. Adams, 1861 is "estuary of the Pei-ho, North China", which is on the coast of the Bohai Sea.One of the localities in this study, Yellow River Estuary, is adjacent to the type locality.Moreover, the shells exhibit remarkable similarity in terms of size, shape, and microsculpture when compared to the descriptions provided by Adams (1861), Yen (1939), Yen (1942), and Kantor and Sysoev (2006), although the available photograph of the holotype is lacking (Agatsuma et al. 2005).We believe that specimens collected in this study belong to common species from the coasts of the Yellow Sea and Bohai Sea in China and are conspecific with the type material.
Native status: Lives on mud bottoms in brackish water areas of estuaries.

Notes:
The genus Assiminea H. & A. Adams, 1865 is cosmopolitan, but most of them are from the Indo-pacific Region. A. estuarina is rarely found in China.The shell of this species is similar to A. hiradoensis Habe, 1942, but there are some differences between them.Each whorl of A. estuarina is more convex than A. hiradoensis.The height of the spire of A. hiradoensis is higher.

Alvania concinna A. Adams, 1861
Diagnosis: Shell minute (4.5±0.41 mm in length, 2.4±0.11mm in width), elongate conical (Fig. 10).Whorls about 6, slightly convex, with elevated spire, deeply sutured.Shell surface reddish-brown, sculptured with many spiral ribs and strong axial ribs; axials tend to weaken on body whorls in populations in temperate waters.Aperture simple, ovate, outer lip thin, without varix, the upper part of outer lip slightly angular.Apex generally not strongly tilted in species with paucispiral protoconch.No umbilicus.
Native status: Lives in intertidal areas in sheltered bays on seaweed.
Distribution: China, Japan and Korea.

Notes:
The trait of the genus Alvania Risso, 1826 is that the teleoconch is ovate-conic to elongate-conic, with clathrate sculpture or spiral sculpture (Adams 1850).The changes of morphological characteristics and surface colour are relatively large, the synonym Hydrobia plicosa E. A. Smith, 1875 is a northern form of this species (Okutani 2017).

Pseudoliotia pulchella Dunker, 1860
Diagnosis: Shell minute (1.3±0.19 mm in length, 3.1±0.16mm in width), depressed conical, round pie, solid (Fig. 11).Whorls about 5, spire very low, body whorl large, the spiral layers are almost in the same plane, whorl periphery appears markedly truncate with a deep concave sulcus between the two peripheral keels.Suture distinct, grooved.Surface sculptured with thick spiral cords and many strong axial ribs, white or covered with yellow crust, thin sculpture lines on the sides of the whorl.Protoconch smooth.Aperture thickened, ovate, declining.Umbilicus deep.
Native status: Under rocks on sandy gravel bottoms in intertidal zones in sheltered areas.
Notes: Pseudoliotia Tate, 1898 is characterised by a depressed conical shell with strong axial and spiral ribs on its surface.P. pulchella is similar to P. astericus, but much larger, lower in shell height and with relatively weak axial ribs.

Peasiella habei D. Reid & Mak,1998
Diagnosis: Shell minute (3.0±0.28 mm in length, 2.4±0.09mm in width), depressed conical, thick, stout; outline domed (Fig. 12).Whorls 3, whorls almost flat-sided or rounded or slightly shouldered; body whorl large with sharply angulate periphery; teleoconch whorls usually smooth, with spiral microstriae.Base flat to slightly rounded, with about 4 spiral ribs.Dark brown with irregular white spots.Suture impressed; peripheral keel prominent, often a projecting flange, rarely slightly undulating.Umbilicus usually narrow; columella narrow, curved at base.Protoconch usually eroded.Aperture ovate, outer lip thin and bottom angular, inner lip slightly thick and the upper part not connected with outer lip.

Native status:
The species is abundant in crevices and amongst barnacles in the middle and upper eulittoral zone, on sheltered and moderately exposed rocky shores; on exposed shores, it shows a preference for surfaces protected from wave action.
Notes: This species is variable in conspicuous features of the shell including colour, spire profile and sculpture, but consistent characteristics are the row of dark spots above the periphery, which extend on to the pale peripheral keel, the darker and often black spire whorls and the prominent keel at the periphery.Pseudoliotia pulchella (Dunker, 1860).
Checklist of the micromolluscs in the intertidal zone of the Yellow Sea ...

Acteocina fusiformis A. Adams, 1850
Diagnosis: Shell minute (2.5±0.15mm in length, 0.9±0.08 mm in width), cylindrical to fusiform, thick, solid, with fine and indistinct spiral grooves (Fig. 13).Surface white, smooth, slightly corroded.Whorls about 3, spire small, body whorl was slightly constricted on the upper part.Shoulder acutely angulated.Protoconch prominently projecting.Suture oblique.Columella stout, with a fold above.slightly convex, the height of the body whorl is larger than spire.Surface covered with yellowish-brown periostracum.Suture distinct, grooved.Growth lines coarse and flexuous.Aperture ovate.Columella with a weak fold.Outer lip margin with a shallow posterior sinus.
Native status: Sucking body fluid of Crassostrea gigas on rocks in intertidal zones.
Distribution: North of China, Japan.

Notes:
The genus Brachystomia Monterosato, 1884 is very similar to Odostomia spp. in shell characters, but differs in the protoconch turning down on the teleoconch apex, attaching to other molluscs when alive (Okutani 2017).
Native status: Sucking body fluid of Crassostrea gigas on rocks in intertidal zones.
Distribution: North of China, Japan.

Turbonilla osyuensus Nomura, 1936
Diagnosis: Shell minute (3.8±0.13 mm in height, 1.0±0.21mm in width), towered, moderately solid, white (Fig. 17).Whorls about 10, each whorl with convex sides.Sutures constricted.Axial ribs oblique and straight, terminating at periphery.Interspaces of axial ribs twice as wide as ribs, the axial rib of the upper whorl is aligned with the interspace of the lower whorl.Aperture simple, upper of the outer lip with angular, columella with no folds, basal lip circle.The apex of the protoconch is composed of two parts of different sizes.Checklist of the micromolluscs in the intertidal zone of the Yellow Sea ...

Native status:
Found on sandy mud bottoms of seagrass beds.
Distribution: Bohai Sea of China, Japan.
Notes: Chemnitzia osyuensis (Nomura, 1936) is the type specimen and a synonym.Very similar to T. gracilenta, but differs in the protoconch rotated right (Higo and Goto 1993).
Native status: Living on seagrass.

Notes:
The shell characteristics of the genus Rissoella are thin; vitreous and soft parts can be seen through it.As some species are similar in morphology, many species are misidentified or undescribed.In this study, our sampled material agrees with the original description of this species (Golikov et al. 1987), as well as the study from Siaden et al. (2019).
Umbo prominent, situated slightly behind the mid-point of the dorsal margin.Break located near posterior margin.Hinge plate very strong, with one cardinal socket, with one anterior lateral cardinal tooth and two posterior lateral cardinal teeth of different sizes in right valve.
Native status: Attached to the byssus of the septifer and the roots of seaweeds between tidal marks.
Distribution: Yellow Bay (Shandong Province) of China, Japan.
Notes: Lasaea consists of small pelecypod species, the largest of which attains a size of about 8 mm in length; adult specimens of the smaller species are about 2 mm long.In general, these shells are quadrangular in outline; they are equivalved, but inequilateral, the anterior end being the longer (Keen 1938).Most, if not all, species of Lasaea are nestlers, finding shelter in dead barnacle tests and amongst the holdfasts of seaweeds.It is cosmopolitan, but only L. undulata is found in China.

Alveinus ojianus Yokoyama, 1927
Diagnosis: Shell minute (1.1±0.17mm in length, 1.0±0.22mm in height), triangularovate, inflated, equivalved (Fig. 20).Umbo prominent, almost situated at the middle of the dorsal margin.Anterior and posterior ends round.Surface smooth and yellowish, ornamented by densely-spaced co-marginal lamellae.Hinge plate with one anterior and one posterior cardinal tooth in the left valve and one cardinal tooth in the right ligament external parivincular, with small resilium.
Native status: Found on sandy mud bottoms in upper sublittoral zones.
Distribution: Yellow Sea and Bohai Sea of China, southern Japan.Native status: Found on sandy mud bottoms in upper sublittoral zones.
Distribution: Bohai Sea of China.
Notes: This species is very similar to Bentharca spp. in shell characters including radial riblets and is covered with lamellate epidermis at interstices, cardinal plate and umbo.

Discussion
This study described the diversity of common micromolluscs from the Yellow Sea and Bohai Sea in China.A total of 20 species were collected, belonging to 16 families and 16 genera of Gastropoda and 3 families and 3 genera of Bivalve, respectively.The statistics show that Caenogastropoda has the highest species diversity, which should be attributed to two essentially micromollusc groups, Rissooidea and Truncatelloidea (Fig. 22A).However, Vetigastropoda have the least diversity.This was due to the micromolluscs of Vetigastropoda being difficult to collect, as many in the families, Solariellidae, Liotinidae, Crosseolidae, Skeneidae, Anatomidae and Scissurellidae inhabited the subtidal zone (10-500 m depth).In terms of morphology, this study analysed the surface characteristics of the shells in detail by taking SEM images of the shells and carried out morphological identification and description, but there are still many undetermined species and, due to the limited amount of sample, a further morphological study could not be carried out.Meanwhile, this also means that there are still many micromolluscs to describe.
According to the survey, it is found that most of the micromolluscs inhabit seagrass (25%), sandy mud bottom (25%), algae (15%), mud and under rocks (5%) and others habitats (respectively 10%) (Fig. 22B).A total of about 40% of them living on seagrass and algae may be associated with their feeding and further refuging habits to reduce predation pressure (Holthuis 1995).Interestingly, B. bipyramidata and O. subangulata from Pyramidellidae were associated with Crassostrea gigas, which are often considered parasites or symbionts of bivalves (Fretter and Graham 1986), while some Pyramidellidae species, such as Odostomia turrita, Odostomia acuta and Spiralinella spiralis, were confirmed to associate with tubeworms from polychaetes (Høisaeter 2014).Some 25% of collected samples were from sandy mud bottoms, because most of them contained only a shell.There are also fewer micromolluscs inhabiting mud flat and rocks, which may indicate that shellfish like to choose the best habitat in terms of habitat space and this selectivity is related to the geography of the sea area.Factors such as location, bottom deposition type, wave disturbance, abundance of food and seasonal changes are closely related.

Distribution:
From Liaoning to Shandong coasts of China; Japan; Australia.Notes: Alaba picta with obvious morphological variance in colour, aperture, shell length and sculpture (Liu 2013).Diala vitrea G. B. Sowerby, 1915 is the name given to a phenotype lacking a colour-pattern (Okutani 2017).Diala picta A. Adams, is the original name of A. picta, which is from the Shandong coast of China (Adams 1861).

Figure 22 .
Figure 22.The statistical chart of micromolluscs diversity and habitat.A The numbers in front of the species names indicate the number of collected specimens.Different colours represent different orders.B The percentage of species in different habitats.Different colours represent different habitats.