Morphological Description and Phylogenetic Analyses of a New Species of Callistoctopus (Cephalopoda, Octopodidae) from China ^†

Abstract

A new octopus species, Callistoctopus tenuipes sp. nov., was formally described from the southeastern coastal waters of China using morphological description and molecular analysis methods. C. tenuipes sp. nov. is a small- to moderate-sized octopus, which is characterized by very narrow and long arms. Although it was previously misidentified as the juvenile of Octopus minor (Sasaki, 1920), it can be recognised by spots, gill lamellae count, funnel organ shape, enlarged suckers, and ligula shape. C. tenuipes sp. nov. differs from the small-sized octopus Callistoctopus xiaohongxu, mainly in the gill lamellae count, funnel organ shape, and arm-length index. In the molecular analysis, sequences obtained from the cytochrome c-oxidase subunit I (COI) gene of eight specimens were 590 bp in length. The pairwise Kimura 2-parameter (K2P) genetic distances between Octopodidae species ranged from 8.58 to 23.79% based on the COI gene. The phylogenetic analyses suggested that C. tenuipes sp. nov. belonged to the Callistoctopus clade and may have a close affinity with C. xiaohongxu and O. minor. Moreover, three species delimitation methods all strongly supported C. tenuipes as a separate species.

1. Introduction

The total length of China’s coastline is over 32,000 km, of which the mainland coastline is approximately 18,000 km and the island coastline is about 14,000 km. The East China Sea and the South China Sea have a high biological diversity and productivity as a consequence of the warm current flowing from the tropics [1]. In the past 20 years, five new species [2,3,4,5], a newly recorded squid [6], and a newly recorded and redescribed octopus Amphioctopus ovulum (Sasaki, 1917) [7] have been reported from Chinese waters. In 2012, Lu, et al. [8] recorded 134 species of cephalopods in Chinese waters. In 2022, Xu, et al. [9], using DNA barcoding analysis, found that there was an underestimation of species diversity in cephalopods in Chinese waters. Therefore, there may be far more than 134 species of cephalopods in Chinese waters, and it is important to continuously review and describe the cephalopods of China.

The genus Callistoctopus is characterized by a reddish body with white spots or stripes and long arms, often regarded as “Octopus macropus group” in the past [10]. Currently, this genus contains 14 members, including Callistoctopus alpheus (Norman, 1993), Callistoctopus aspilosomatis (Norman, 1993), Callistoctopus bunurong (Stranks, 1990), Callistoctopus dierythraeus (Norman, 1993), Callistoctopus graptus (Norman, 1993), Callistoctopus lechenaultii (d’Orbigny, 1826), Callistoctopus luteus (Sasaki, 1929), Callistoctopus macropus (Risso, 1826), Callistoctopus nocturnus (Norman and Sweeney, 1997), Callistoctopus ornatus (Gould, 1852), Callistoctopus rapanui (Voss, 1979), Callistoctopus xiaohongxu Zheng, Xu and Li, 2022, Callistoctopus furvus (Gould, 1852), and Callistoctopus taprobanensis (Robson, 1926), with the status of C. furvus and C. taprobanensis unresolved [11]. In the last few decades, C. furvus has been considered a potential name for the “macropus” of the West Atlantic. However, Jesus, et al. [12] recently separated this species from C. macropus and used integrative methods of ethnoknowledge, classic taxonomy, and molecular analysis to describe a neotype. Thus, two species of Callistoctopus are now known to exist in the Mediterranean Sea and the Atlantic Ocean, whereas the other members of the genus are scattered throughout the Indo-Pacific region. In the Indo-Pacific waters, several informally described species of this genus have been reported in recent years. Three unverified species of Callistoctopus from the coast of Vietnam were briefly described by Kaneko, et al. [13] and labeled as Callistoctopus sp.1, Callistoctopus sp.2, and Callistoctopus sp.3. Meanwhile, Sreeja [14] found two cryptic Callistoctopus species along the coast of Kerala, India.

The diversity and taxonomy of cephalopods in China have been studied over the last 20 years [9,15,16,17]. The new cryptic Callistoctopus species described in this study was first collected in Xiamen, Fujian Province, and then reported in a DNA-barcoding study of cephalopods from Chinese waters in 2011 [15]. In subsequent sample collections, we were fortunate to have collected this octopus species several times on Dongshan Island in Fujian Province. This new species and another similar octopus, C. xiaohongxu, which was reported in this area, are both small in size, with reddish-orange skin and long arms. Octopus minor (Sasaki, 1920) is a common economic species with red skin and long arms in East Asia, and these two small octopuses were misidentified as juveniles of O. minor on Dongshan Island. This new species and C. xiaohongxu were called Xiaohongxu by the locals in Chinese, which means small and red-skinned octopus.

In this study, this new octopod species is formally described and named Callistoctopus tenuipes sp. nov. Phylogenetic trees were reconstructed using COI gene sequences to analyse the phylogenetic position of C. tenuipes sp. nov in the family Octopodidae.

2. Materials and Methods

2.1. Specimen Collection

All samples were collected from Dongshan Fish Market, Fujian Province, China. After collecting, samples were covered with ice, then fixed in 10% formalin for 7 days, and then preserved in 70% alcohol. The type specimens are deposited in the Specimen Room, Fisheries College, Ocean University of China (OUC), China.

2.2. Morphological Analysis

Measurements, counts, and indices follow Roper and Voss [18], Norman and Sweeney [19], and Huffard and Hochberg [20]. Abbreviations and definitions are as follows: ALI—arm length index (arm length/ML × 100); AWI—arm width index (arm width/ML × 100); CaLI—calamus length index (calamus length/ligula length × 100); DWDI—the deepest web depth index (the deepest web depth/the longest arm × 100); EgC—egg count; EgL—egg length; EgW—egg width; FFLI—free funnel length index (free funnel length/funnel length × 100); FLI—funnel length index (funnel length/ML × 100); GC—gill count (number of gill lamellae per outer demibranch, excluding the terminal lamella); HAMI—hectocotylised arm index (hectocotylised arm length/ML × 100); HASC—hectocotylised arm sucker count; HWI—head width index (head width/ML × 100); LLI—ligula length index (ligula length/hectocotylised arm length × 100); ML—dorsal mantle length; MWI—mantle width index (mantle width/ML × 100); OAI—opposite arm index (hectocotylised arm length/normal third arm length × 100); PAI—pallial aperture index (pallial aperture length/ML × 100); SC—sucker count on normal arms; SDIn—normal sucker diameter index (normal sucker diameter/ML × 100); SpC—spermatophore count; SpL—spermatophore length; SpW—spermatophore width; SWDI—the shallowest web depth index (the shallowest web depth/the longest arm × 100); TL—total length; TW—total wet weight.

The beaks and radulae were removed from the buccal mass of some specimens, then cleaned and stored in 75% ethanol. The radulae were cleaned with 10% NaOH, air dried, covered with gold, and then scanned using a VEGA3 scanning electron microscope.

2.3. DNA Extraction, PCR Amplification, and Sequencing

Total genomic DNA was extracted from muscle tissue using CTAB (Hexadecyl trimethyl ammonium bromide) method [21]. DNA was dissolved in TE buffer (10 mM Tris-HCI, 1 mM EDTA, pH 8.0). The COI fragments were amplified by primers referenced by Folmer, et al. [22]. The amplification was carried out in 50 µL reactions, containing 1 µL template DNA of 100 ng, 2 µL of each primer (10 µM), 25 µL of 2 × Hieff^® PCR Master Mix (With Dye) (Yeasen), and 20 µL sterile distilled water. The PCRs were performed under the following conditions: denaturation at 94 °C for 5 min, 34 cycles at 94 °C for 10 s, annealing at 55 °C for 20 s, and extension at 72 °C for 30 s, and a final extension of 72 °C for 5 min. PCR products were sequenced with the Sanger sequencing method.

2.4. Molecular and Phylogenetic Analyses

SeqMan v.7.2 (DNASTAR package) was used to assemble sequences, which were then deposited in GenBank under accession number OP184666-OP184673. The other sequences were downloaded from GenBank (Table 1). All sequences were aligned using the Clustal W in MEGA X [23]. Vampyroteuthis infernalis Chun, 1903 was used as an outgroup. The Kimura 2-parameter (K2P) model was used to generate K2P distances in MEGA X [23] as well.

The nucleotide substitution model was selected based on the Bayesian information criterion (BIC) in ModelFinder [24]. The best-fit model GTR + G4 + I + F was selected to construct the BI tree in MrBayes 3.2.6 [25]. The Bayesian analysis was started with four default heat chains, running 1.2 million generations of the Markov Chain Monte Carlo (MCMC) until the standard deviation of split frequencies value was lower than 0.01. The first 25% of generations were discarded as burn-in while the remaining 75% was used to construct the phylogenetic tree. Maximum likelihood phylogenies were inferred using IQ-TREE [26] under the model automatically selected for 1000 ultrafast [27] bootstraps, as well as the Shimodaira–Hasegawa-like approximate-likelihood ratio test [28].

Table 1. Information of specimens for the COI gene used in this study.

Species	GenBank Accession No.	Locality	References
Amphioctopus aegina	NC_029702	Haikou, Hainan, China	[29]
Amphioctopus fangsiao	HQ846126	Xiamen, Fujian, China	[15]
Amphioctopus marginatus	NC_036351	Haikou, Hainan, China	[30]
Amphioctopus neglectus	NC_049899	Nanning, Guangxi, China	[31]
Amphioctopus rex	MN987271	Coastal water of India	[7]
Callistoctopus aspilosomatis	AB430525	Miyagi Island, Okinawa, Japan	[32]
Callistoctopus furvus	MT892962	Morro de São Paulo, Bahia, Brazil	[12]
Callistoctopus luteus	MT214050	Zhangzhou, Fujian, China	[9]
Callistoctopus macropus	MN933634	Mediterranean Sea	[33]
Callistoctopus ornatus	HM104257	-	[34]
Callistoctopus tenuipes	OP184666-OP184673	Zhangzhou, Fujian, China	This study
Callistoctopus xiaohongxu	OP135961	Zhangzhou, Fujian, China	[5]
Callistoctopus sp.1	AB385875	Nha Trang, Vietnam	[32]
Callistoctopus sp.2	AB385876	Nha Trang, Vietnam	[32]
Callistoctopus sp.3	AB385877	Nha Trang, Vietnam	[32]
Callistoctopus sp.4	KF489435	Kerala, India	[14]
Callistoctopus sp.5	MN933632	Coastal water of Brazil	[33]
Cistopus chinensis	KF017606	Xiamen, Fujian, China	[35]
Cistopus taiwanicus	HQ846142	Xiamen, Fujian, China	[15]
Octopus cyanea	NC_039847	Xisha Islands, China	Unpublished
Octopus minor 1	NC_015896	Weihai, Shandong, China	[36]
Octopus minor 2	AB191275	Akashi, Osaka, Japan	[37]
Octopus minor 3	MF631967	Penghu Islands, China	[16]
Octopus sinensis	OK001740	Wenzhou, Zhejiang, China	[38]
Octopus vulgaris	KU525762	Galicia, Spain	[39]
Vampyroteuthis infernalis	NC_009689	Ogasawara Island, Japan	[40]

Table 1. Information of specimens for the COI gene used in this study.

Species	GenBank Accession No.	Locality	References
Amphioctopus aegina	NC_029702	Haikou, Hainan, China	[29]
Amphioctopus fangsiao	HQ846126	Xiamen, Fujian, China	[15]
Amphioctopus marginatus	NC_036351	Haikou, Hainan, China	[30]
Amphioctopus neglectus	NC_049899	Nanning, Guangxi, China	[31]
Amphioctopus rex	MN987271	Coastal water of India	[7]
Callistoctopus aspilosomatis	AB430525	Miyagi Island, Okinawa, Japan	[32]
Callistoctopus furvus	MT892962	Morro de São Paulo, Bahia, Brazil	[12]
Callistoctopus luteus	MT214050	Zhangzhou, Fujian, China	[9]
Callistoctopus macropus	MN933634	Mediterranean Sea	[33]
Callistoctopus ornatus	HM104257	-	[34]
Callistoctopus tenuipes	OP184666-OP184673	Zhangzhou, Fujian, China	This study
Callistoctopus xiaohongxu	OP135961	Zhangzhou, Fujian, China	[5]
Callistoctopus sp.1	AB385875	Nha Trang, Vietnam	[32]
Callistoctopus sp.2	AB385876	Nha Trang, Vietnam	[32]
Callistoctopus sp.3	AB385877	Nha Trang, Vietnam	[32]
Callistoctopus sp.4	KF489435	Kerala, India	[14]
Callistoctopus sp.5	MN933632	Coastal water of Brazil	[33]
Cistopus chinensis	KF017606	Xiamen, Fujian, China	[35]
Cistopus taiwanicus	HQ846142	Xiamen, Fujian, China	[15]
Octopus cyanea	NC_039847	Xisha Islands, China	Unpublished
Octopus minor 1	NC_015896	Weihai, Shandong, China	[36]
Octopus minor 2	AB191275	Akashi, Osaka, Japan	[37]
Octopus minor 3	MF631967	Penghu Islands, China	[16]
Octopus sinensis	OK001740	Wenzhou, Zhejiang, China	[38]
Octopus vulgaris	KU525762	Galicia, Spain	[39]
Vampyroteuthis infernalis	NC_009689	Ogasawara Island, Japan	[40]

2.5. Species Delimitation

We implemented three widely used molecular methods for species delimitation, including Automatic Barcode Gap Discovery (ABGD), Generalized Mixed Yule Coalescent model (GMYC), and Bayesian Poisson Tree Processes (bPTP). The ABGD analysis was performed on the online server (https://bioinfo.mnhn.fr/abi/public/abgd/) using Kimura (K80) distance model with the relative gap width (X) set to 1.0 and the other parameters maintained as default [41]. The GMYC model was conducted in the split package in R using single-threshold and multiple-threshold analyses (https://www.R-project.org/) [42], respectively. GTR + G4 + I + F was selected as the substitution model, which was evaluated by ModelFinder [24]. The ultrametric tree was generated in BEAST 2.7.1 with a Yule model and strict clock as an assumption [43], running 30 million generations and sampling every 10,000 generations. Then Tracer v1.7.2 was used to assess the convergence by effective sample size (ESS) of each value [44]. The bPTP analysis was carried out on the bPTP online server (https://species.h-its.org/ptp/) with 10,000 generations and 0.1 burn-in [45].

3. Results

3.1. Molecular and Phylogenetic Analyses

Fragments obtained from eight specimens were 590 bp in length. The K2P genetic distances between C. tenuipes sp. nov. and the other species in Octopodidae based on the COI gene are shown in

Table 2. Pairwise Kimura 2-parameter (K2P) genetic distance (%) between Octopodidae species analysed in this study based on the COI gene.

	A. a	A. f	A. m	A. n	A. r	Ca. a	Ca. f	Ca. l	Ca. m	Ca. o	Ca. t	Ca. x	Ca. sp.1	Ca. sp.2	Ca. sp.3	Ca. sp.4	Ca. sp.5	Ci. c	Ci. t	O. c	O. m 1	O. m 2	O. m 3	O. s
A. a	-
A. f	14.40	-
A. m	11.05	14.67	-
A. n	14.11	16.24	12.65	-
A. r	11.87	16.46	13.95	11.84	-
Ca. a	20.25	20.27	20.76	22.96	21.28	-
Ca. f	20.20	20.97	21.20	21.95	20.00	12.78	-
Ca. l	20.36	19.95	21.54	20.97	19.95	15.32	15.19	-
Ca. m	19.54	20.51	20.29	21.49	19.82	12.32	3.39	15.47	-
Ca. o	20.97	20.31	20.53	20.53	20.58	11.03	4.75	16.27	5.05	-
Ca. t	20.27	20.54	20.09	23.79	18.86	13.15	12.40	15.00	11.08	11.09	-
Ca. x	18.43	19.22	19.26	21.10	18.50	14.02	11.69	15.74	12.12	11.92	8.58	-
Ca. sp.1	18.65	17.28	19.70	20.90	19.69	14.85	13.89	15.76	13.83	12.10	11.26	11.89	-
Ca. sp.2	21.26	19.95	22.10	23.60	20.84	12.30	13.03	13.95	13.45	13.24	12.53	12.59	12.55	-
Ca. sp.3	21.00	17.78	21.33	21.58	21.10	12.53	14.15	15.67	13.21	12.78	11.65	11.70	12.12	3.91	-
Ca. sp.4	19.32	19.38	20.07	22.04	21.55	10.83	12.60	14.70	11.71	10.86	10.42	10.44	13.17	6.02	5.63	-
Ca. sp.5	20.01	20.77	20.76	21.74	19.82	12.74	0.00	15.19	3.32	4.85	12.37	11.67	13.61	13.20	14.31	12.56	-
Ci. c	18.19	18.73	18.96	22.57	19.16	21.58	21.77	21.12	19.86	20.39	19.38	18.00	19.87	21.23	18.51	18.50	21.55	-
Ci. t	16.36	18.94	17.53	20.13	17.51	20.04	18.33	21.46	18.65	17.72	21.43	19.34	19.62	19.39	19.38	18.65	17.96	13.21	-
O. c	15.07	16.88	15.78	18.06	18.05	17.11	17.19	17.72	17.75	17.08	17.07	15.33	19.13	19.91	18.70	18.73	17.29	17.09	16.15	-
O. m 1	19.80	19.37	19.82	23.74	21.74	13.61	11.75	14.44	10.86	11.52	10.28	11.07	13.60	14.60	13.90	11.97	11.72	18.43	20.77	17.51	-
O. m 2	20.98	20.54	20.76	24.46	22.70	14.92	12.60	15.62	11.69	12.35	10.89	11.91	14.69	15.71	15.00	13.03	12.56	19.58	21.72	18.19	2.00	-
O. m 3	19.10	19.12	20.07	22.73	20.28	13.40	11.96	15.19	10.23	10.45	8.82	10.23	12.73	13.71	13.02	11.32	11.93	18.20	21.00	17.27	2.38	3.70	-
O. s	14.42	18.05	15.95	18.54	18.52	21.01	21.49	21.00	20.28	20.29	20.30	20.54	17.99	20.79	20.80	20.08	21.28	17.60	17.85	17.13	20.74	21.94	20.02	-
O. v	14.37	17.51	18.01	17.50	18.29	21.51	21.00	21.04	20.14	20.15	19.90	20.95	16.44	21.84	20.71	20.43	21.00	18.87	18.60	18.30	20.69	22.08	19.31	2.97

Abbreviations: A. a, Amphioctopus aegina; A. f, Amphioctopus fangsiao; A. m, Amphioctopus marginatus; A. n, Amphioctopus neglectus; A. r, Amphioctopus rex; Ca. a, Callistoctopus aspilosomatis; Ca. f, Callistoctopus furvus; Ca. l, Callistoctopus luteus; Ca. m, Callistoctopus macropus; Ca. o, Callistoctopus ornatus; Ca. t, Callistoctopus tenuipes; Ca. x, Callistoctopus xiaohongxu; Ca. sp.1, Callistocotpus sp.1; Ca. sp.2, Callistocotpus sp.2; Ca. sp.3, Callistocotpus sp.3; Ca. sp.4, Callistocotpus sp.4; Ca. sp.5, Callistocotpus sp.5; Ci.c, Cistopus chinensis; Ci. t, Cistopus taiwanicus; O. c, Octopus cyanea; O. m 1, Octopus minor 1; O. m 2, Octopus minor 2; O. m 3, Octopus minor 3; O. s, Octopus sinensis; and O. v, Octopus vulgaris.

, ranging from 8.58 to 23.79%.

The COI gene dataset represented four genera of Octopodidae. The Maximum-Likelihood tree (Figure 1) showed that Octopodidae were divided into two clades. One clade comprised the Callistoctopus species and O. minor, with a bootstrap probability (BP) of 84%. The other clade included Octopus, Amphioctopus, and Cistopus. Eight specimens formed a highly supported monophyletic clade, with a BP of 95%. C. tenuipes sp. nov. had a close relationship with C. xiaohongxu and O. minor group, whereas the BP value was ≤ 50%. The topology of the Bayesian Inference analysis is presented in Figure 2. Similarly, C. tenuipes sp. nov. fell into a monophyletic branch with a posterior probability (PP) of 1. C. tenuipes sp. nov., C. xiaohongxu, and O. minor group converged into a single clade. In both phylogenetic trees, species of the genus Amphioctopus and Cistopus formed a monophyletic clade, respectively. However, Octopus was observed to be polyphyletic.

3.2. Species Delimitation

The results of the three species delimitation methods of ABGD, GMYC, and bPTP are shown in Figure 1. Eight specimens of C. tenuipes sp. nov. were recognised as a separate molecular operational taxonomic unit (MOTU) by all three methods. ABGD and bPTP methods showed similar results, with a total of 18 MOTUs identified. GMYC retrieved 25 and 22 MOTUs for the single-threshold model and multiple-threshold model, respectively.

3.3. Taxonomy

Order Octopoda Leach, 1818.

Family Octopodidae d’Orbigny, 1840.

Genus Callistoctopus Taki, 1964.

Type species: Callistoctopus ornatus (Gould, 1852).

Callistoctopus tenuipes sp. nov. (Figure 3, Figure 4, Figure 5 and Figure 6).

Holotype. OUC-201812050307, mature male, 37.6 mm ML, 23.42° N, 117.85° E, 5 December 2018, coll.

Paratypes. OUC-201812050308, mature male, 41.4 mm ML, 23.42° N, 117.85° E, 5 December 2018, coll. OUC-201812050309, mature male, 42.8 mm ML, 23.42° N, 117.85° E, 5 December 2018, coll. OUC-201812050310, mature male, 41.1 mm ML, 23.42° N, 117.85° E, 5 December 2018, coll. OUC-201808200302, mature male, 30.5 mm ML, 23.42° N, 117.85° E, 20 August 2018, coll. OUC-201812050311, mature female, 54.7 mm ML, 23.42° N, 117.85° E, 5 December 2018, coll. OUC-201812050312, mature female, 57.3 mm ML, 23.42° N, 117.85° E, 5 December 2018, coll. OUC-201812050313, immature female, 34.6 mm ML, 23.42° N, 117.85° E, 5 December 2018, coll.

Diagnosis: Small- to medium-sized species. Mantle ovoid to elongate. Stylets absent. Arms long and slender, with dorsal arms longest (arm formula 1 > 2 > 3 > 4). Web shallow, web formula typically A > B > C > D > E. Suckers small and biserial, enlarged suckers absent. Hectocotylised arm with 67–73 suckers. Ligula moderate size (around 5% of hectocotylised arm length), triangular with shallow groove. Funnel organ W-shaped. Gills with 8 to 10 lamellae per demibranch. Colour typically reddish-orange to reddish-brown in fresh specimens. Lateral margins of arms start with two lines of chromatophores under the skin, then increase to three rows and end in a single line.

Description: The following description is based on five mature males and three females (two mature, one immature). Counts, measurements, and indices are presented in

Table 3. Measurements (mm), counts, and morphometric indices of Callistoctopus tenuipes sp. nov.

Catalogue Number	OUC-201812050307	OUC-201812050308	OUC-201812050309	OUC-201812050310	OUC-201808200302	OUC-201812050311	OUC-201812050312	OUC-201812050313
Status	Holotype	Paratype	Paratype	Paratype	Paratype	Paratype	Paratype	Paratype
Sex	♂	♂	♂	♂	♂	♀	♀	♀
Maturity	Mature	Mature	Mature	Mature	Mature	Mature	Mature	Immature
TL	232.6	318.8	216.8	214.4	146.5	284.0	283.4	156.1
TW(g)	7.9	11.3	11.6	9.3	12.3	27.3	20.6	8.2
ML	37.6	41.4	42.8	41.1	30.5	54.7	57.3	34.6
MWI	52.4	59.4	60.7	48.7	95.1	56.7	58.3	60.7
PAI	72.3	101.1	63.1	67.4	87.5	72.5	71.6	69.7
HWI	30.6	26.8	30.8	36.7	40.7	25.6	30.2	31.5
AL1I (L/R)	500.3/478.5	635.7/D	388.8/377.6	395.1/373.7	D/401.3	385.4/401.3	D/364.7	D/263.6
AL2I (L/R)	356.6/345.5	473.9/439.4	331.5/304.4	273.5/319.7	379.3/D	331.3/318.4	D/322.7	322.3/332.9
AL3I (L/R)	359.6/186.4	419.1/192.3	293.7/154.4	298.1/148.9	D/193.8	235.7/230.7	264.0/D	278.0/273.1
AL4I (L/R)	310.4/299.7	345.2/D	D/D	D/280.5	335.4/334.8	211.8/225.6	254.4/247.8	D/253.5
AWI	11.4	12.1	13.8	11.4	20.7	13.2	13.4	15.9
DWDI	A:8.7	A:6.8	A:10.9	A:9.7	A:13.8	A:9.2	A:8.6	A:10.2
SWDI	E:3.5	E:3.9	E:6.5	E:5.0	E:9.0	E:7.1	E:6.0	E:6.9
FLI	29.0	38.9	31.1	31.1	31.5	38.7	30.9	27.7
FFLI	53.2	58.4	46.6	38.3	55.2	44.8	47.5	33.3
SDIn	4.8	4.8	4.4	4.4	6.2	3.4	3.3	4.0
HAMI	186.4	192.3	154.4	148.9	193.8	-	-	-
OAI	51.8	45.9	52.6	50.0	D	-	-	-
HASC	68H	73H	68H	67H	72H	-	-	-
LLI	6.3	5.2	7.2	4.4	6.8	-	-	-
CaLI	34.1	24.4	30.4	44.4	35.0	-	-	-
GC	8/9	9/9	8/8	8/8	8/8	9/10	8/9	8/9
SpC	-	4	-	-	-	-	-	-
SpL	-	59.9	-	-	-	-	-	-
SpW	-	1.6	-	-	-	-	-	-
EgC	-	-	-	-	-	114	111	-
EgL	-	-	-	-	-	13.7	15.1	-
EgW	-	-	-	-	-	4.2	4.5	-

D: Damaged.

. Small- to moderate-sized species (ML 30.5–57.3 mm). Total length 146.5–318.8 mm. Body weight to 27.3 g. Skin smooth. Mantle ovoid to elongate. Mantle opening length moderate (PAI 63.1–101.1). Head width narrower than mantle width (HWI 25.6–40.7). Funnel short (FLI 27.7–38.9), free funnel length around 33–58% funnel length (FFLI 33.3–58.4). Funnel organ W-shaped (Figure 4D), outer limbs approximately equal in length with medial ones. Arms long (ALI 148.9–635.7) and slender (AWI 11.4–20.7), dorsal arms longest (typical arm formula 1 > 2 > 3 > 4). Webs shallow (WDI 3.5–13.8) (Figure 4B). Dorsal webs deepest, ventral webs shallowest (web formula A > B > C > D > E).

Suckers in two rows, small-sized (SDIn 3.3–6.2) (Figure 4B), enlarged suckers absent. The third right arm of mature males hectocotylised with 67–73 suckers, approximately 50% length of the opposite arm (OAI 45.9–52.6). Ligula small, triangular with shallow groove (Figure 4C), about 5% of arm length (LLI 4.4–7.2). Calamus of moderate size (CaLI 24.4–44.4). Gills with 8–10 lamellae per demibranch.

Digestive tract (Figure 5A): Buccal mass moderate size. Anterior salivary glands small. Posterior salivary glands large, triangular, and as long as buccal mass. Crop well developed. Stomach about as large as caecum. Caecum with about one whorl. Intestine long. Digestive gland well developed, dark grey. Ink sac present, embedded in digestive gland, and attached to the intestine posteriorly, then opening into anus. Anal flaps small (Figure 4E).

Chitinous beaks dark brown. Upper beak (Figure 5B) with short hood, sharp and small rostrum. Crest curved and longer than total wing length. Lower break (Figure 5C,D) with a medium-sized hood, moderate broad wings, and short lateral wings. Radula (Figure 5E,F) with seven teeth and two marginal plates per transverse row. Rachidian teeth with two or three lateral cups on each side. First lateral teeth small and sharp. Second lateral teeth larger than the first, sharp. Third lateral teeth long, curved, sharply pointed, longer than second lateral teeth. Marginal plates flat.

Male reproductive tract (Figure 6A): Terminal organ ‘—’ shaped (Figure 6B) in mature males. Spermatophore gland long. Accessory gland curved, almost same length as the Needham’s sac. Spermatophore storage sac long and slender. Vas deferens short, narrow. Testis roundish and large. Spermatophores narrow (Figure 6C), average length 46.4 mm, few (4–6 in storage sac).

Female reproductive tract (Figure 6D–F): Ovary large and round in mature females. Proximal oviducts short and narrow. Distal oviducts long and thin. About 110 eggs in ovary of a single mature female. Largest mature ovarian eggs approximately 15.1 mm.

Colouration: Fresh specimens reddish orange. Skin smooth with no papillae on dorsal mantle. Two or more lines of chromatophores obviously presented on the lateral margins of arms under the skin (Figure 4A).

Etymology: The species name tenuipes is proposed for its very narrow arms.

Distribution: Through the survey of fishermen and our sampling collection, this species is mainly found in the East China Sea to the South China Sea, and potentially south to Hainan Province, including Beibu Gulf.

Remarks: The species reported in this study have differences between males and females. Generally, mature females are larger than males. To determine the maturation times, we collected samples three times from the same sea area and found that males and females mature synchronously.

4. Discussion

As mentioned above, C. tenuipes sp. nov. was misidentified as the juvenile of O. minor. To prove the validity of the nominal taxon C. tenuipes sp. nov., we compared it with the original descriptive features of three unsolved subspecies of O. minor.

Table 4. Comparison of Callistoctopus tenuipes sp. nov., Callistoctopus xiaohongxu, and three subspecies of Octopus minor group.

Item	Callistoctopus tenuipes sp. nov.	Callistoctopus xiaohongxu	Octopus minor typicus	Octopus minor pardalis	Octopus minor minor
Data source	This study	[5]	[46]	[46]	[47]
Body weight	to 27.3 g	to 39.2 g	-	-	-
Skin	smooth, reddish-orange	smooth, reddish-orange to reddish-brown	smooth, red-brown	slightly firm, dark brown in alcohol	slightly warty, grey brown
Spots	no spot	no spot	light yellow spots on mantle surface	five transverse streaks on mantle surface	irregular marbled stripe
GC	8–10	8–9	10–12	14	7–9
Funnel organ	W-shaped	\ ∧ /-shaped	V V-shaped	U U-shaped	V V-shaped
WDI	3.5–13.8	15.7–22.9	deepest around 10	-	deepest around 12
ALI	247.8–635.7	154.9–336.3	400–500	around 400–700	around 300–700
Enlarged suckers	absent	absent	present	-	-
Ligula	triangular with shallow groove, LLI 4.4–7.2	cylindrical with groove, LLI 7.0–11.6	spoon-like with wide hollow groove, LLI around 18–23	-	-

shows that C. tenuipes sp. nov. differs from the other three subspecies, mainly in spots, gill count, funnel organ shape, enlarged suckers, and ligula shape. Meanwhile, C. tenuipes sp. nov. is distinct from C. xiaohongxu in body size (mature individuals to 39.2 g versus 27.3 g), funnel organ shape (W-shaped versus \ ∧ /-shaped), web depth index (WDI 3.5–13.8 versus 15.7–22.9), arm length index (ALI 247.8–635.7 versus 154.9–336.3), and ligula index (LLI 4.4–7.2 versus 7.0–11.6). On the other hand, C. tenuipes sp. nov. resembles C. xiaohongxu in orange skin as in the fish market. However, in mature individuals, C. tenuipes sp. nov. has a smaller body size and longer arms than C. xiaohongxu. Notably, chromatophores on the lateral margins of arms under the skin were also found in O. minor and C. xiaohongxu. However, the arrangement of chromatophores in the three species was not consistent. In addition, small chromatophores of an unresolved octopus Callistoctopus sp.1, collected by Kaneko, et al. [13] from Vietnamese waters, were scattered on the surface of the arms. It remains to be seen whether chromatophores will be found in more species and whether there is a pattern of arrangement of chromatophores in certain taxa.

In the molecular analysis, the pairwise K2P genetic distances displayed in Table 3 indicate that C. tenuipes sp. nov. can be separated as a new species since the genetic distance between other sequences of Octopodidae analysed in this study ranged from 8.58 to 23.79%. Three species delimitation analyses also strongly supported C. tenuipes sp. nov. as a separate new species. Phylogenetic topologies showed that C. tenuipes sp. nov. may have a close relationship with C. xiaohongxu and O. minor. Regarding the taxonomic status of O. minor, Kaneko, et al. [32] placed it into Callistoctopus based on its morphological characteristics and DNA-barcoding analyses. In this study, three O. minor sequences converged to the Callistocotpus clade, which may indicate that O. minor had a closer affinity to the genus Callistocotpus than Octopus. However, the taxonomic classification of O. minor still needs further study.

In summary, we described a new octopod species, C. tenuipes sp. nov., using morphological and molecular methods. The South China Sea coastal current flows from Guangdong Province, and one branch flows southwest along the Hainan coast. Considering the current system, we speculate that this new octopus will be collected in Hainan and Beibu Gulf after a comprehensive resource survey. Our work adds some useful information to the classification of octopuses. Moreover, this study increases the knowledge of species diversity in the Southeast China Sea.