A New Record and Three Redescriptions of Rissoinidae from China’s Hainan Island, with the First Presentation of Two Mitochondrial Genomes in the Family Rissoinidae

Abstract

The family Rissoinidae represents a significant component of microgastropod diversity, with a global distribution spanning temperate to tropical zones and encompassing over 300 recorded species. Hainan Island, the largest island in the South China Sea, harbors a rich diversity of mollusks, but the family Rissoinidae remains poorly studied in this region. Here, we report three rissoinid species and one newly recorded species from Hainan Island, providing detailed taxonomic descriptions supported by SEM imaging. For the first time, we provide the mitochondrial genomes of Rissoina cardinalis and Phosinella seguenziana, analyzing their genome structure and nucleotide composition, thereby addressing the existing knowledge gap in Rissoinidae research. A phylogenetic tree of the family Rissoinidae was reconstructed using the COI gene, clarifying the intergeneric relationships within the family. Notably, the genus Rissoina is revealed as a non-monophyletic group, likely due to the limitations of single-gene analyses in providing adequate phylogenetic information.

1. Introduction

Marine microgastropods represent a significant yet underexplored component of gastropod diversity [1]. The family Rissoinidae W. Stimpson, 1865 (Rissooidea) constitutes one of the predominant microgastropod families, encompassing over 300 extant species distributed globally. Members of this family are widely distributed, spanning temperate to tropical regions [2]. These gastropods predominantly inhabit shallow waters, with the highest species diversity observed in the low intertidal and shallow sublittoral zones, where algae, stones, or corals provide refuge. The family Rissoinidae possesses a well-documented fossil record, tracing back to at least the Middle Jurassic period [3]. Research on rissoinids has historically been constrained, predominantly focusing on systematics [2,3,4].

Owing to the pronounced convergence in shell morphology, rissoinids have undergone a complex and evolving classification process. Rissoinids were initially classified within the family Rissoidae, with certain groups designated as the subfamily Rissoininae [5,6]. Taxonomists classified groups with an apophysis on the inner side of the horny operculum as part of the family Rissoinidae, which was later demoted to a subfamily within Rissoidae [7,8,9,10]. Since then, the questions regarding the systematic classification of this group has remained unresolved. Bouchet et al. [11] elevated the subfamily Rissoininae to the rank of family Rissoinidae, a change supported by phylogenetic evidence and widely accepted by scholars. To date, Rissoinidae comprises 37 extant genera and 13 fossil genera [12]. Research efforts have predominantly focused on the Mediterranean, South Pacific, and Atlantic Oceans [13,14,15,16]. However, there is a notable lack of research on the taxonomy and phylogeny of Rissoinidae in the South China Sea.

Hainan Island, the largest island in the South China Sea, harbors a remarkable diversity of mollusks. This study conducted a comprehensive survey of the diversity and taxonomy of Rissoinidae on Hainan Island and reconstructed the molecular phylogeny of the family. Additionally, this study is the first to report and characterize two complete mitochondrial genomes of Rissoinidae. This work offers novel insights into the diversity and evolutionary relationships of Rissoinidae in the South China Sea.

2. Materials and Methods

2.1. Taxon Sampling and Processing

Samples were collected from the intertidal zone of Hainan Island on 20 September 2022 and 10 September 2024. All specimens were preserved in 95% ethanol. Standard measurements were performed using a stereomicroscope equipped with an eyepiece micrometer. DNA extraction was performed on intact specimens using the commercial TIANamp Marine Animals DNA Kit (Tiangen Biotech, Beijing, China). All procedures strictly followed the manufacturer’s protocol. The extracted DNA was maintained at −4°C for short-term preservation. Shells, radulae, and opercula were examined using a scanning electron microscope (SEM) (TESCAN (China) Co., Ltd., Shanghai, China), following the protocols described by Xu et al. [17] and Qi et al. [18]. For SEM studies of the radulae, the surrounding tissues were digested with proteinase K during DNA extraction using the TIANamp Marine Animals DNA Kit. Radulae were precipitated to the bottom of the centrifuge tube after separation and collected using a pipette. Then, the residual material at the bottom of the centrifuge tube was transferred onto a glass slide using a micropipette, and the radula was carefully relocated to a clean slide under a stereomicroscope. Subsequently, the radulae were washed with drops of distilled water or 10% KOH on glass histology slides. The shells, radulae, and opercula were affixed to stubs, coated with a layer of gold via sputtering, and examined using a TESCAN VEGA3 scanning electron microscope (TESCAN (China) Co., Ltd., Shanghai, China).

2.2. Sequencing, Assembly, and Annotation

Total genomic DNA was subjected to library preparation and 150 bp paired-end sequencing using Illumina’s HiSeq X platform, with all technical procedures conducted at Beijing Novogene Technology Co., Ltd. (Beijing, China). The bioinformatics analysis followed the pipeline described by Qi et al. [19]. The clean sequencing reads were de novo assembled through SPAdes v3.13.0 [20], employing the software’s standard parameter settings. The complete mitochondrial genome was reconstructed using NOVOPlasty 4.3.5 [21], with the previously published COI gene of Rissoina ambigua as the seed. The mitogenomes were annotated through the MITOS WebServer [22]. The complete circular visualization of the mitochondrial genome was constructed by employing CGView, a specialized tool for mitogenome mapping [23]. The AT and GC skew values were computed according to the standard formulae: AT skew = (A − T)/(A + T), and GC skew = (G − C)/(G + C).

2.3. Phylogenetic and Genetic Divergence Analysis

As no mitochondrial genomes of Rissoinidae were available in GenBank, we reconstructed phylogenetic trees for the family based on COI sequences, combining our data with GenBank sequences, which included six Rissoinidae taxa and one Barleeidae species, Ansola angustata (Pilsbry, 1901), as the outgroup (Table 1). COI genes of Rissoina cardinalis Brazier, 1877 and Phosinella seguenziana (Issel, 1869) from the family Rissoinidae were extracted from their mitochondrial genomes. Phylogenetic analyses were conducted using the maximum likelihood (ML) method. ML analyses were performed using IQ-TREE [24] with the nucleotide substitution model K3Pu+F+G4 selected by ModelFinder, along with 1000 ultrafast bootstrap replicates. The pairwise distances of COI within the Rissoinidae clade were calculated using the p-distance in MEGA X [25].

Table 1. GenBank accession numbers for specimens included in the molecular analyses.

Family	Species	Genbank
Rissoinidae	Phosinella seguenziana (Issel, 1869)	PQ767087
	Rissoina ambigua (A. Gould, 1849)	MW277843
	Rissoina cardinalis Brazier, 1877	PQ767088
	Rissoina sp._PNG1679	MZ539691
	Rissoina sp._USNM_1466845	MZ559526
	Pandalosia subulata Laseron, 1956	AB930474
	Stosicia annulata (Dunker, 1860)	AB930480
Barleeiidae	Ansola angustata (Pilsbry, 1901)	AB930479

Note: Bold labels indicate mitochondrial genomes obtained in this study, while other sequences represent cox1 genes.

3. Results and Discussion

3.1. Systematics

Rissoinidae W. Stimpson, 1865

Rissoina d’Orbigny, 1841

Type species: Rissoa inca A. d’Orbigny, 1841 from the delta of Peru.

Rissoina cardinalis Brazier, 1877

Costalynia birestes Laseron, 1956

Rissoina (Rissolina) cardinalis Brazier, 1877

Type locality: Papua New Guinea.

Description: Shell—minute (6.2 ± 0.04 mm in height; 1.3 ± 0.02 mm in width), narrowly conical, stout; most specimens are uniformly white, but some exhibit a pale-brown spiral band on the abapical half of the spire whorls and the adapical half of the body whorl (Figure 1A,B). Protoconch of planktotrophic larval type, of 1.5, relatively weakly convex whorls, each being 1.5 times wider than high; protoconch–teleoconch boundary sharply demarcated, with moderately thickened margin (Figure 1G,H). Teleoconch of 7.5 very weakly convex whorls; spire whorls weakly angulate below and above suture; body whorl weakly angulate below suture and strongly contracted near the base; suture weakly undulating, not impressed (Figure 1C,D). Axial ribs of prominent, very narrow, rather distantly spaced, rounded to sharp, weakly opisthocline ribs; interspaces rather deep and slightly wider than axial ribs (Figure 1F). Spiral sculpture is absent on spire whorls, apart from some very weak and irregularly spaced lirae; last whorl with a rather variable number of spiral riblets—usually two or three—above contraction near shell base; body whorl with prominent, narrow, and nodular spiral fold, which is ornamented with fine, inconspicuous lirae (Figure 1F). Aperture D-shaped; inner lip thin, apart from moderately prominent swelling near rather wide, shallow, and short anterior channel; anterior margin of anterior channel moderately curved dorsally; inner side of outer lip thin; prominent and moderately wide labial varix, bearing very prominent spiral ribs on its entire length or restricted on abapical half; outer lip very weakly opisthocline in profile (Figure 1C,D). No umbilicus.

Figure 1. (A–J) Rissoina cardinalis Brazier, 1877. (A,B) shell illustration in color; (C) scanning electron micrographs of frontal view of shell; (D) scanning electron micrographs of lateral view of shell; (E,F) detail of teleoconch surface; (G) scanning electron micrographs of apical view of protoconch; (H) scanning electron micrographs of lateral view of protoconch; (I) inner face of operculum; (J) outer face of operculum.

Operculum: Horny, thick, yellowish, translucent, D-shaped, posteriorly broadly angled, anteriorly rounded; nucleus eccentric, located in the lower left corner; growth line is obvious, arc-shaped, and radiating; there is a strong, curved, grooved nail-like structure growing from the core on the inner side, and the spike-like structure ends in a V shape; a weak ridge runs parallel to the medial edge (Figure 1I,J).

Radula: Radular teeth interlocked moderately in unfolded condition (Figure 2A–E). Central tooth ${\displaystyle \frac{3-5+1+3-5}{1 1}}$, with long triangular cutting edge, small cusps, and a single pair of basal denticles (Figure 2B); the U-shaped ventral extension area is well developed. Lateral teeth 5 − 6 + 1 + 5 − 6, wide; small cusps at the end, larger primary cusp long and wide; smaller pointed denticles at the sides, 5–6 each inner and outer (Figure 2A). Two rows of marginal teeth, elongated; inner marginal teeth 9 − 12 + 1 + 1 − 2, larger primary cusp, 9–12 cusps on outer, 1–2 on inner; outer marginal teeth 3 − 4 + 1 + 0, only 3–4 cusps on outer (Figure 2C–E).

Figure 2. (A–E) scanning electron micrographs of the radula of Rissoina cardinalis Brazier, 1877. (A) partial photo of radula; (B) central tooth; (C,E) inner marginal teeth; (D) outer marginal teeth.

Distribution: Queensland (Lindeman Island) to Northern Territory (Point Charles) in Australia; southern Papua New Guinea (Katow); and China (Eman Town, Danzhou City, Hainan Island, and Taiwan Province).

Remarks: Rissoina cardinalis Brazier, 1877 strongly resembles Rissoina (Rissolina) angasii Pease, 1871 in shell shape and sculpture, but differs essentially in protoconch structure. R. cardinalis is a planktotrophic larvae type, while R. angasii is a non-planktotrophic larvae type. Furthermore, the outer lip exhibits a prominent and moderately wide labial varix.

Rissoina ambigua (A. Gould, 1849)

Pyramidella ambigua A. Gould, 1849

Rissoina (Rissoina) ambigua (A. Gould, 1849)

Type locality: Clermont Tonnerre Island of Polynesia.

Description: Shell—minute (5.5 ± 0.07 mm in height; 2.3 ± 0.05 mm in width), elongated conical, stout; white with light reddish-brown spiral stripes, 8 whorls (Figure 3A,B). Protoconch of planktotrophic larval type, of 2.5 whorls; of 1 whorl, smooth; of 2–2.5 whorls, weak spiral sculpture; protoconch–teleoconch boundary sharply demarcated (Figure 3H,I). Teleoconch of 6 weakly convex whorls; only axial ribs, interspace slightly wider than axial rib; spiral sculpture absent, apart from some very weak and irregularly spaced lirae (Figure 3C–F). Aperture thin, D-shaped; anterior channel shallow and short, posterior angulated; inner lip thin, smooth, central concave; outer lip thin, outer side of the outer lip without labial varix; axial ribs contract near shell base; no columella (Figure 3G).

Figure 3. (A–J) Rissoina ambigua (A. Gould, 1849). (A,B) Shell illustration in color; (C) scanning electron micrographs of frontal view of shell; (D) scanning electron micrographs of lateral view of shell; (E,F) detail of teleoconch surface; (G) detail of aperture; (H) scanning electron micrographs of lateral view of protoconch; (I) scanning electron micrographs of apical view of protoconch; (J) inner face of operculum.

Operculum: thick, solid, D-shaped, posterior angulated; inner edge with groove, nail-like structure growing from the core on the inner side (Figure 3J).

Distribution: Rissoina ambigua is a widely distributed and abundant species primarily inhabiting the tropical Indian and western Pacific Oceans. Its range extends from Tanzania and Mozambique in the Indian Ocean to the Marquesas and Pitcairn Islands in the east. It is also found in the subtropical and tropical waters of China, Japan, and southwestern Australia, as well as in regions such as the Red Sea and Hawaii.

Remarks: The protoconch of R. ambigua suggests its planktotrophic larval development, typical of species inhabiting subtropical and tropical intertidal zones. It was frequently misidentified as the common species Rissoa rosea Deshayes, 1863, which subsequent taxonomic revisions have reassigned to the genus Rissoina. R. ambigua differs from R. rosea via its larger size and greater color variability, confirming that they are distinct species.

R. ambigua bears a strong resemblance to Rissoina stricta (Menke, 1851), a common species in the Panamic faunal province, particularly in teleoconch characteristics. Both are quite variable in size, slenderness, and number and density of the axial ribs, which resulted in many synonyms. They might be synonyms; however, “the protoconch is somewhat more conical in R. stricta … the penes … differ markedly” [26]. Since there is no information on its anatomy, we provisionally regard this single Clipperton specimen as R. stricta [5].

Apataxia Laseron, 1956

Type species: Apataxia erecta Laseron, 1956 = Rissoina miltozona Tomlin, 1915 from Australia.

Apataxia cerithiiformis (Tryon, 1887)

Rissoina balteata Pease, 1870

Rissoina cerithiiformis “Dunker” Tryon, 1887

Apataxia erecta Laseron, 1956

Type locality: Red Sea.

Description: Shell—minute (2.9 ± 0.03 mm in height; 1.2 ± 0.01 mm in width), thick, rather stout, elongated conical, white, translucent, with yellowish or bright-brown spiral band above the suture line, 9 whorls (Figure 4A–C). Protoconch of planktotrophic larval type, yellowish-brown, 3.5 convex, smooth apart from wavy sculptures in the lower part of the last convex, protoconch–teleoconch boundary clear (Figure 4G,H). Teleoconch of 5.5 weakly convex to almost flat whorls; axial ribs prominent, narrow, interspaces wider than axial ribs, microscopic (only observable through SEM) striae in interspaces between axial ribs (Figure 4D,F), with weak ribs; different whorl have different number of ribs; sutures distinct, grooved; the axial ribs of body whorl contract near shell base; without umbilicus. Aperture small, thick; anterior channel shallow and short, posterior angulated; inner lip smooth, central concave; 4 weak ridges on the inner side of the outer lip.

Figure 4. (A–H) Apataxia cerithiiformis (Tryon, 1887). (A) Shell illustration in color; (B) scanning electron micrographs of frontal view of shell; (C) scanning electron micrographs of lateral view of shell; (D,F) detail of teleoconch surface; (E) detail of aperture; (G) scanning electron micrographs of lateral view of protoconch; (H) scanning electron micrographs of apical view of protoconch.

Distribution: A. cerithiiformis is a widely distributed and common Indo-Pacific species, recorded from the Red Sea and Tanzania to China, Japan, Queensland, and the Hawaiian Islands. There are no previous eastern Pacific records [5]. It has a protoconch indicating planktotrophic larval development.

Remarks: Apataxia Laseron, 1956, initially proposed by Laseron (1956), was subsequently classified as a subgenus of Rissoina by Ponder [9]. Faber and Kaiser [5] reclassified Apataxia as a full genus, rather than a subgenus of Rissoina d’Orbigny, 1840. The former differs from Rissoina in that it does not have an incrassate outer lip; instead, the adult aperture is internally reinforced with a series of spiral ribs ending in tooth-like structures. Consequently, the adult aperture is narrower than that of the sub-adult stage. A. cerithiiformis is a widespread and common species occurring throughout the Indo-Pacific region. Rissoina balteata Pease, 1869, is sometimes considered synonymous with A. cerithiiformis. However, no type material is known [27], and the species is described as having a varicose outer lip [5]. This characteristic does not align with the diagnostic features of the genus Apataxia.

Phosinella Mörch, 1876

Type species: Rissoina pulchra (C. B. Adams, 1850) from Jamaica

Phosinella seguenziana (Issel, 1869)

Rissoina seguenziana Issel, 1869

Rissoina tornatilis A.A. Gould, 1861

Type locality: the Gulf of Suez.

Description: Shell—minute (3.7 ± 0.02 mm in height; 1.6 ± 0.04 mm in width), thick, solid, turret-like, with an elongated spire, white or yellowish, translucent, whorls: 7.5 (Figure 5A–D). Protoconch of planktotrophic larval type, yellowish, about 2.5 convex, smooth, protoconch–teleoconch boundary clear (Figure 5I,J). Teleoconch of 5 weakly convex whorls, spire whorls weakly angulate below suture; the axial ribs and spiral ribs on the shell surface are interlaced in a clathrate sculpture, and the intersection points are nodular; fine spiral sculptures in each gird; 3 ribs on 1-3 whorls of teleoconch, 4 ribs on 5 whorl of teleoconch, 4 ribs on body whorl; the axial ribs of body whorl contract near shell base; single weak spiral near the shell base; sutures moderately deep (Figure 5C,D,F–H). Aperture D-shaped; anterior channel shallow and short, posterior angulated; inner lip thin, smooth, central concave; outer lip thick, slopes forward; the outer side of the outer lip with labial varix; no columella (Figure 5E).

Figure 5. (A–L) Phosinella seguenziana (Issel, 1869). (A,B) Shell illustration in color; (C) scanning electron micrographs of frontal view of shell; (D) scanning electron micrographs of lateral view of shell; (E) detail of aperture; (F–H) detail of teleoconch surface; (I) scanning electron micrographs of lateral view of protoconch; (J) scanning electron micrographs of apical view of protoconch; (K) outer face of operculum; (L) inner face of operculum.

Operculum: Horny, thick, yellow, D-shaped; nucleus eccentric, located in the lower left corner; growth line is obvious; there is a strong, curved, grooved nail-like structure growing from the core on the inner side, and the spike-like structure ends in a V shape; a weak ridge runs parallel to the medial edge (Figure 5K,L).

Distribution: Red Sea, Indian Ocean, and tropical Western Pacific as far east as Fiji and northward to China’s Hainan Island and Beibu Gulf.

Remarks: The genus Phosinella differs from Rissoina primarily in its clathrate sculptured shell, while remaining similar in other aspects of shell morphology. Phosinella is predominantly distributed in the tropical Indo-Western Pacific region. P. seguenziana strongly resembles P. media (Schwartz von Mohrenstern, 1860) in shell shape and sculpture, but differs in that it has single weak spiral folds near the shell base instead of two or three prominent folds in P. media. In this study, P. seguenziana is recorded in China for the first time.

3.2. Mitogenome Architecture Genome

The mitogenomes of Rissoina cardinalis (Figure 6, Genbank PQ767088) and Phosinella seguenziana (Figure 7, Genbank PQ767087) are 17321bp and 17221bp, respectively. Each comprises 37 genes, including 13 PCGs, 2 rRNAs genes, 22 tRNAs genes, and a putative control region (CR). The CRs are 605bp and 666bp, respectively. The total lengths of the concatenated 13 PCGs of Rissoina cardinalis and Phosinella seguenziana are 11310bp and 11403bp, respectively, with an average A + T contents of 63.72% and 63.57% (Table 2 and Table 3). ATG is the most frequently used start codon; however, nad4 uses ATA in Rissoina cardinalis (Table 4), while cox3, nad2, and nad4 use ATA and TTA in Phosinella seguenziana (Table 5). The terminal codons of Rissoina cardinalis and Phosinella seguenziana consist of TAG and TAA, respectively. The total lengths of the tRNA sequences in R. cardinalis and P. seguenziana are 1541 bp and 1545 bp, with an A + T contents of 67.16% and 66.34%, respectively. The rrnL and rrnS genes are 1562 bp and 1199 bp in size, with an A + T contents of 68.56% and 69.22% in R. cardinalis, and 1635 bp and 1135 bp, with an A + T contents of 67.62% and 64.84% in P. seguenziana (Table 2 and Table 3), respectively.

Figure 6. Map of the complete mitochondrial genome of Rissoina cardinalis Brazier, 1877.

Figure 7. Map of the complete mitochondrial genome of Phosinella seguenziana (Issel, 1869).

Table 2. The nucleotide composition of the Rissoina cardinalis mitogenome.

Genes or Regions	Size	Nucleotide Composition				A + T	AT Skew	GC Skew
		T	C	A	G	(%)
Complete mitogenome	17,321	39.26	12.34	26.63	21.76	65.89	−0.19	0.27
PCGs	11,310	40.91	13.48	22.81	22.79	63.72	−0.28	0.25
tRNA genes	1541	34.52	13.82	32.64	19.01	67.16	−0.03	0.16
rRNA genes	2761	34.77	10.54	34.08	20.61	68.85	−0.01	0.32
lrRNA	1562	35.85	10.50	32.71	20.93	68.56	−0.05	0.33
SrRNA	1199	33.36	10.59	35.86	20.18	69.22	0.04	0.31
A + T-rich region	605	40.56	6.71	38.38	14.35	78.94	−0.03	0.36

Table 3. The nucleotide composition of the Phosinella seguenziana mitogenome.

Genes or Regions	Size	Nucleotide Composition				A + T	AT Skew	GC Skew
		T	C	A	G	(%)
Complete mitogenome	17,221	37.88	12.89	27.25	21.98	65.13	−0.16	0.26
PCGs	11,403	40.26	13.83	23.31	22.6	63.57	−0.27	0.24
tRNA genes	1545	33.01	14.37	33.33	19.29	66.34	0.005	0.15
rRNA genes	2770	33.03	11.19	34.04	21.73	67.07	0.015	0.32
lrRNA	1635	34.37	10.64	34.25	20.73	67.62	−0.002	0.32
SrRNA	1135	31.10	11.98	33.74	23.17	64.84	0.04	0.32
A + T-rich region	666	34.49	8.7	38.99	17.83	73.48	0.06	0.34

Table 4. Annotated mitochondrial genome of Rissoina cardinalis.

Gene	Direction	Position		Size	Intergenic	Condon		Anti-Codon
		From	TO		Nucleotides	Start	Stop
cox1	F	1	1536	1536	—	ATG	TAG	—
cox2	F	1552	2238	687	15	ATG	—	TTT
atp8	F	2330	2485	156	91	ATG	—	TGG
trnD	F	2533	2604	72	47	—	—	TCG
atp6	F	2618	3313	696	13	ATG	—	GTT
trnG	R	3420	3490	71	106	—	—	GAT
trnQ	R	3527	3604	78	36	—	TAG	—
trnC	R	3620	3685	66	15	—	—	GCT
trnW	R	3707	3776	70	21	—	TAG	—
trnY	R	3812	3879	68	35	—	TAG	—
trnM	R	3932	4004	73	52	—	TAA	—
trnE	R	4009	4076	68	4	—	TAG	—
rrnS	F	4077	5275	1199	0	—	—	GTC
trnV	F	5276	5344	69	0	—	TAG	—
rrnL	F	5345	6906	1562	0	—	—	TCC
trnL2	F	6907	6976	70	0	—	—	TTG
trnL1	F	7008	7075	68	31	—	—	GCA
nad1	F	7076	8020	945	0	ATG	—	TCA
trnP	F	8108	8175	68	87	—	—	GTA
nad6	F	8179	8697	519	3	ATG	—	CAT
cob	F	8752	9891	1140	54	ATG	—	TTC
trnS2	F	9894	9960	67	2	—	—	—
trnF	F	9976	10045	70	15	—	—	TAC
trnT	R	10084	10152	69	38	—	—	—
nad4l	F	10220	10498	279	67	ATG	—	TAA
nad4	F	10507	11877	1371	8	ATA	—	TAG
trnH	F	11958	12025	68	80	—	TAA	—
nad5	F	12027	13745	1719	1	ATG	—	TGG
cox3	F	14351	15133	783	605	ATG	TAA	—
trnK	F	15191	15260	70	57	—	TAG	—
trnA	F	15290	15357	68	29	—	—	TGA
trnR	F	15416	15493	78	58	—	—	GAA
trnN	F	15540	15608	69	46	—	—	TGT
trnI	F	15620	15690	71	11	—	TAA	—
nad3	F	15716	16045	330	25	ATG	TAG	—
trns1	F	16063	16132	70	17	—	—	GTG
nad2	F	16136	17284	1149	3	ATG	TAA	—

Table 5. Annotated mitochondrial genome of Phosinella seguenziana.

Gene	Direction	Position		Size	Intergenic	Condon		Anti-Codon
		From	To		Nucleotides	Start	Stop
cox1	F	1	1554	1554	—	ATG	883	—
cox2	F	1588	2277	690	33	ATG	—	TTT
atp8	F	2364	2519	156	86	ATG	—	TGG
trnD	F	2545	2616	72	25	—	—	TCG
atp6	F	2659	3393	735	40	ATG	—	GTT
trnY	R	3460	3526	67	66	—	—	GAT
trnC	R	3528	3598	71	1	—	TAA	—
trnW	R	3616	3687	72	17	—	—	GCT
trnQ	R	3716	3781	66	28	—	TAA	—
trnG	R	3838	3906	69	56	—	TAA	—
trnM	R	3948	4016	69	41	—	TAG	—
trnE	R	4032	4102	71	15	—	TAA	—
rrnS	F	4103	5237	1135	0	—	—	GTC
trnV	F	5238	5307	70	0	—	TAG	—
rrnL	F	5308	6942	1635	0	—	—	GTA
trnL2	F	6943	7020	78	0	—	—	GCA
trnL1	F	7120	7185	66	99	—	—	TCA
nad1	F	7190	8134	945	4	ATG	—	TTG
trnP	F	8177	8247	71	42	—	—	TCC
nad6	F	8256	8768	513	8	ATG	—	CAT
cob	F	8791	9930	1140	22	ATG	—	TTC
trnS2	F	9930	9995	66	−1	—	—	—
trnF	F	10002	10084	83	6	—	—	TAC
trnT	R	10090	10153	64	5	—	—	—
nad4l	F	10189	10479	291	35	ATG	—	TAA
nad4	F	10482	11855	1374	2	TTA	—	TAG
trnH	F	11866	11932	67	10	—	TAA	—
nad5	F	11936	13663	1728	3	ATG	—	TGG
cox3	F	14330	15133	804	666	ATA	TAA	—
trnK	F	15183	15258	76	49	—	TAA	—
trnA	F	15266	15332	67	7	—	—	TGA
trnR	F	15344	15414	71	11	—	—	GAA
trnN	F	15428	15495	68	13	—	—	TGT
trnI	F	15515	15585	71	19	—	TAG	—
nad3	F	15589	15942	354	3	ATG	TAA	—
trns1	F	15959	16028	70	16	—	—	GTG
nad2	F	16064	17182	1119	35	ATA	TAA	—

3.3. Phylogenetic and Genetic Diversity Analysis

The phylogenetic analysis of the family Rissoinidae, including most Rissoinidae species with COI in the NCBI, inferred the phylogenetic relationships of rissoinids (Figure 8). Pandalosia forms a strongly supported sister group with Stosicia. Rissoina does not form a monophyletic group. However, Phosinella and Rissoina sp. cluster together to form a branch, which in turn forms an unsupported sister group with Rissoina ambigua. Although Rissoina cardinalis represents the outermost branch within the family Rissoinidae, it is unsupported. The non-monophyly of Rissoina is further corroborated by Francesco et al. [28] and may be attributed to the limitations of single-gene analyses in providing a sufficient phylogenetic signal. Pairwise genetic distances among species of Rissoinidae, derived from the COI gene, are presented in Table 6. Pairwise genetic distances within the family Rissoinidae range from 2.9% (Rissoina sp._PNG1679 vs. Rissoina sp._USNM_1466845) to 33.4% (Rissoina cardinalis vs. Pandalosia subulata). Genetic distances between genera range from 16.1% (Phosinella seguenziana vs. Rissoina ambigua) to 33.4% (Pandalosia subulata vs. Rissoina cardinalis). Genetic distances within the genus Rissoina range from 2.9% to 22.9%.

Figure 8. Summary tree from maximum likelihood analysis of COI sequences. Taxa highlighted in blue represent specimens newly sequenced in this study. Ansola angustata (Barleeiidae) was selected as the outgroup.

Table 6. Pairwise p-distance among species of Rissoinidae (%).

Species	1	2	3	4	5	6
Phosinella seguenziana	-
Rissoina ambigua	16.1	-
Rissoina cardinalis	21.5	19.7	-
Rissoina sp._PNG1679	17.7	17.2	22.8	-
Rissoina sp._USNM_1466845	17.2	16.9	22.9	2.9	-
Pandalosia  subulata	32.1	31.0	33.4	30.4	31.5	-
Stosicia annulata	28.1	25.6	29.7	28.8	27.7	29.9

4. Conclusions

Based on SEM examination, we report four species of Rissoinidae from Hainan Island, China, revealing detailed morphological characteristics of the shells, protoconchs, and opercula, among which Phosinella seguenziana represents a new record for China. The phylogenetic relationships of Rissoinidae were reconstructed using the COI gene, revealing that the genus Rissoina is not monophyletic, likely due to the limitations of single-gene analyses in providing sufficient phylogenetic signals. This study presents the first two complete mitochondrial genomes of Rissoinidae, offering foundational data for future research to resolve the phylogenetic relationships within the family.