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Research Article
Morphological description and mitochondrial DNA-based phylogenetic placement of a new species of Callistoctopus Taki, 1964 (Cephalopoda, Octopodidae) from the southeast waters of China
expand article infoXiaodong Zheng‡, Chenxi Xu‡, Jiahua Li‡
‡ Ocean University of China, Qingdao, China

Corresponding author: Xiaodong Zheng ( xdzheng@ouc.edu.cn )

Academic editor: Pavel Stoev
© 2022 Xiaodong Zheng, Chenxi Xu, Jiahua Li.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Zheng X, Xu C, Li J (2022) Morphological description and mitochondrial DNA-based phylogenetic placement of a new species of Callistoctopus Taki, 1964 (Cephalopoda, Octopodidae) from the southeast waters of China. ZooKeys 1121: 1-15. https://doi.org/10.3897/zookeys.1121.86264
ZooBank: urn:lsid:zoobank.org:pub:18A5CC36-D9F4-488A-AB4D-E7C30179839A
Open Access

Abstract

In this study, we described a new species of octopus and named it Callistoctopus xiaohongxu sp. nov. based on nine specimens captured in the waters of southeast China. Callistoctopus xiaohongxu sp. nov. is a small to moderate-sized octopus. The most characteristic and defining morphological features are the reddish-orange to reddish-brown skin, gills with 8 or 9 lamellae per demibranch, \∧/-shaped funnel organ, and small suckers. Fragments obtained from the mitochondrial cytochrome c oxidase subunit I (COI) gene of nine specimens were 593 bp in length, and the genetic distance among the specimens of C. xiaohongxu sp. nov. and the other 16 octopods ranged from 11.13 to 21.09%. Topologies resulting from ML and BI analyses of the COI gene showed a highly supported monophyletic clade (bootstrap value [BS] = 94%, posterior probability [PP] = 100%) containing all the specimens identified as C. xiaohongxu sp. nov.

Keywords

Callistoctopus xiaohongxu sp. nov., COI gene, new species, octopus, taxonomy

Introduction

Among the cephalopods, 134 species have been recorded in the China Seas (Li 1983; Dong 1988; Zheng et al. 1999; Lu et al. 2012).Due to the influence of three strong warm currents – the Kuroshio Current (KC), the South China Sea Current (SCSC), and the Taiwan Current (TC) – water temperatures of the East China Sea and South China Sea range between 14–16 °C in coastal areas even during winter (Liu 2013), providing ideal environmental conditions to generate abundant marine biodiversity, as well as cephalopods.

Species in the genus Callistoctopus were previously treated as the “Octopus macropus group”, from which Norman (1993) separated four new species. The current taxonomy of species in this genus is mainly based on morphological features, while there are still very limited molecular data. In Chinese waters, only two species Callistoctopus ornatus (Gould, 1852) and C. luteus (Sasaki, 1929), have been recorded so far (Lu et al. 2012; Norman et al. 2014).

In this paper, we described one new species of Callistoctopus, which was called ‘xiaohongxu’ in Chinese for its smooth skin and reddish-brown colour, from the southeast China Sea area. The newly discovered species has been mistakenly identified and sold in fish markets of Dongshan Island in Zhangzhou, Fujian Province, as juveniles of ‘Octopus’ minor (Sasaki, 1920). However, based on the obvious differences in the size of the adult animals, gill lamellae number, and the funnel organ shape, we can readily distinguish this new species from ‘O.’ minor externally. Here we present a full morphological description and genetic analyses of the new species of octopod.

Materials and methods

Specimen collection

Samples were collected from Dongshan Seafood Market Pier (23°25'12"N, 117°51'0"E) in Zhangzhou, Fujian Province, China. The type specimens are deposited in the Specimen Room, Fisheries College, Ocean University of China (OUC), Qingdao, China. All specimens were attributed to mature or immature stages based on the absence or presence of spermatophores in males, and ovary fullness or egg development in females.

Morphological feature analyses

All specimens were measured after being fixed according to Roper and Voss (1983) and indices were calculated on the basis of Huffard and Hochberg (2005). Abbreviations: TL – total length; ML – mantle length; WF – web formula (web sectors ordered from deepest to shallowest); GC – gill count (number of gill lamellae per outer demibranch, excluding the terminal lamella); SC – number of suckers on normal arms; MWI – mantle width index (mantle width/ML×100).; HWI – head width index (head width/mantle width×100); WDI – the web depth index (deepest web length/longest arm×100); ALI – arm length index (arm length/ML×100); AWI – arm width index (arm width/ML×100); SDI – sucker diameter index (sucker diameter/ML×100); FLI – funnel length index (funnel length/ML×100); FFLI – free funnel length index (free funnel length/funnel length×100); PAI – pallial aperture index (pallial aperture/mantle width×100); LLI – ligula length index (ligula length/hectocotylized arm length×100); CaLI – calamus length index (calamus length/ligula length×100); HAMI – hectocotylized arm mantle index (hectocotylized arm length/ML×100); OAI – opposite arm index (hectocotylized arm length/normal third arm length×100); HASC – number of suckers on hectocotylized arm of male; SpC – spermatophore count; SpL – spermatophore length; SpW – spermatophore width; EgC – egg count; EgL – egg length; EgW – egg width. All measurements are in millimeters and weights in grams.

The beaks and radulae were removed from the buccal mass. Then beaks were cleaned and stored in 75% ethanol. Seven beak morphological indices, upper hood length (UHL), upper crest length (UCL), upper rostrum length (URL), upper rostrum width (URW), lower hood length (LHL), lower crest length (LCL), and lower rostrum width (LRW), were measured to the nearest 0.01 mm by Vernier caliper (Clarke, 1986). Five ratios were calculated as follows: UHL/UCL, URW/UCL, URL/UHL, LHL/LCL, and LRW/LCL. The radulae were cleaned with 10% NaOH, air-dried, coated with gold, and then scanned using a VEGA3 scanning electron microscope. Funnel organ and anal flaps were stained with methylene blue.

DNA extraction and sequencing

Before fixation with formalin and alcohol, about 100 mg of muscle tissue was cut from the mantle inside all chilled specimens. Total genomic DNA was extracted using a CTAB (hexadecyltrimethylammonium bromide) method (Winnepenninckx 1993). DNA was dissolved in TE buffer (10 mM Tris-HCI, 1 mM EDTA, pH 8.0) and stored at –30° C. Regions of mitochondrial cytochrome c oxidase subunit I (COI) fragments were amplified using primers referenced to Octopus conispadiceus Sasaki, 1917 by Ma et al. (2016). These sequences were amplified by PCR with the following conditions: 94 °C (3 min), 34 cycles of 94 °C (45 s), 50 °C (1 min), 72 °C (1 min), and a final extension of 72 °C (5 min).

Molecular analyses

The COI sequences of the other 17 species were downloaded from GenBank (Table 1). Vampyroteuthis infernalis Chun, 1903 was used as an outgroup in all analyses. ModelFinder (Kalyaanamoorthy et al. 2017) was used to select the best-fit model using the BIC criterion. Maximum likelihood phylogenies were inferred using IQ-TREE (Nguyen et al. 2015) under the GTR+I+G4+F model for 1000 ultrafast (Minh et al. 2013) bootstraps, as well as the Shimodaira-Hasegawa-like approximate likelihood-ratio test (Guindon et al. 2010). Bayesian inference phylogenies were inferred using MrBayes 3.2.6 (Ronquist et al. 2012) under GTR+I+G4+F model (4 parallel runs, 1 000 000 generations) as well, in which the initial 25% of sampled data were discarded as burn-in. And COI sequences of new species have been deposited in GenBank under accession numbers OP135961-OP135969. Pairwise comparisons of the distances based on COI gene were also calculated by MEGA X under the Kimura 2-parameter model (Kumar et al. 2018).

Table 1.
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GenBank accession numbers for species analysed in this study.

Species GenBank numbers References
Amphioctopus aegina NC_029702 Zhang et al. (2017)
Amphioctopus fangsiao HQ846126 Dai et al. (2012)
Amphioctopus neglectus MH899749 Tang et al. (2019)
Amphioctopus rex MF447874 Tang et al. (2019)
Callistoctopus ornatus HM104257 Strugnell et al. (2014)
Callistoctopus aspilosomatis AB430525 Kaneko et al. (2011)
Callistoctopus luteus NC_039848 Unpublished
Callistoctopus macropus MN933634 Lima et al. (2020)
Callistoctopus xiaohongxu OP135961-OP135969 This study
Cistopus chinensis KF017606 Cheng et al. (2013)
Cistopus taiwanicus NC_023257 Cheng et al. (2013)
Octopus vulgaris KU525762 Amor et al. (2017)
Octopus bimaculatus NC_028547 Dominguez-Contreras et al. (2016)
Octopus conispadiceus KJ789854 Ma et al. (2016)
Octopus cyanea NC_039847 Unpublished
‘Octopus’ minor HQ638215 Cheng et al. (2012)
Octopus sinensis MT712046 Li et al. (2021)
Vampyroteuthis infernalis NC_009689 Yokobori et al. (2007)

Results

Taxonomy

Order Octopoda Leach, 1818

Family Octopodidae d’Orbigny, 1840

Callistoctopus Taki, 1964

Type species

Callistoctopus ornatus (Gould, 1852).

Callistoctopus xiaohongxu sp. nov.

https://zoobank.org/C4E08679-59A2-47AF-AA85-D19D1F78415B
Figs 1, 2, 3, 4

Type material

Holotype : OUC-201808200301, mature ♂, 45.5 mm ML, Dongshan Seafood Market Pier, Zhangzhou, Fujian, China, 20 August 2018, coll. Paratypes: OUC-201812050301, mature ♂, 49.5 mm ML, Dongshan Seafood Market Pier, Zhangzhou, Fujian, China, 5 December 2018, coll. OUC-201812050302, mature ♂, 53.2 mm ML, Dongshan Seafood Market Pier, Zhangzhou, Fujian, China, 5 December 2018, coll. OUC-201812050303, mature ♂, 56.3 mm ML, Dongshan Seafood Market Pier, Zhangzhou, Fujian, China, 5 December 2018, coll. OUC-201806080302, immature ♀, 50.7 mm ML, Dongshan Seafood Market Pier, Zhangzhou, Fujian, China, 8 June 2018, coll. OUC-201812050305, mature ♀, 51.7 mm ML, Dongshan Seafood Market Pier, Zhangzhou, Fujian, China, 5 December 2018, coll. OUC-201812050306, mature ♀, 83.3 mm ML, Dongshan Seafood Market Pier, Zhangzhou, Fujian, China, 5 December 2018, coll.

Figure 1. 

Callistoctopus xiaohongxu sp. nov., holotype, male, 45.5 mm ML (OUC-201808200301) A photograph of dorsal view B photograph of ventral view. Scale bars: 10 mm (A, B).

Other material

OUC-201812050304, mature ♂, 63.2 mm ML, Dongshan Seafood Market Pier, Zhangzhou, Fujian, China, 5 December 2018, coll. OUC-201806080301, immature ♀, 41.7 mm ML, Dongshan Seafood Market Pier, Zhangzhou, Fujian, China, 8 June, 2018, coll.

Diagnosis

Small to moderate size (ML 41.7–83.3 mm). Colour of skin reddish-orange to reddish-brown, no papillae or patch. One or two lines of black chromatophores on the lateral margins of arms under the skin (Fig. 2A). Head narrow (HWI 23.0–39.1). Arms of moderate length (ALI 154.9–336.3), thin (AWI 8.7–18.0). Web deep (WDI 15.7–22.9). Suckers small (SDI 5.0–6.9) and biserial. Enlarged suckers absent. Funnel organ \ /\ /-shaped, long (FLI 51.0–68.5). Gills with 8–9 lamellae per demibranch. Ligula moderate size (LLI 7.0–11.6) with groove.

Figure 2. 

Callistoctopus xiaohongxu sp. nov. A proximal portion of arms 1–3 (left side), male, 49.5 mm ML (OUC-201812050301) B funnel organ, male, 53.2 mm ML (OUC-201812050302) C oral view of basal portion of arms, male, 63.2mm ML (OUC-201812050304) D distal portion of hectocotylus, male, 63.2 mm ML (OUC-201812050304). Abbreviations: c, calamus; l, ligula. Scale bars: 10 mm (B, C, D).

Description

Measurements and indices of nine specimens are presented in Table 2. Small to moderate-size species (ML 41.7–83.3 mm), total length (TL) 195.7–382.1 mm, body weight up to 39.2 g. Skin smooth, one or two lines of black chromatophores on the lateral margins of arms under the skin (Fig. 2A). Mantle slightly ovoid to elongate, muscular. Head width narrower than mantle width (HWI 23.0–39.1). Stylets absent. Funnel long (FLI 51.0–68.5), free funnel length around 24–46% funnel length (FFLI 23.9–46.0), funnel organ \∧/-shaped (Fig. 2B). Outer limbs slightly shorter than medial limbs. Arms moderate length (ALI 154.9–336.3), slender (AWI 8.7–18.0), dorsal arms always longest (arm formula of most specimens belongs to 1 > 2 > 4 > 3). Suckers in two rows (Fig. 2C), small (SDI 5.0–6.9). In larger animals, 157–198 suckers on each normal arm, and the first or second arm has the most suckers. Enlarged suckers absent. Webs deep (WDI 15.7–22.9), typical web formula A > B > C > D > E. The third right arm of mature males hectocotylized, length approximately 60–80% of the opposite arm (Fig. 2D). Ligula of moderate size, robust and cylindrical with deep groove. LLI ranges from 7.0–11.6 of arm length. Calamus of moderate size, around 25–30% of ligula length (CaLI 26.3–31.6). Hectocotylized arm with 70–83 suckers. Gills with 8–9 lamellae per demibranch.

Table 2.
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Measurements (mm) and indices for Callistoctopus xiaohongxu sp. nov. Abbreviation: D, damaged.

Name OUC-201808200301 OUC-201812050301 OUC-201812050302 OUC-201812050303 OUC-201812050304 OUC-201806080301 OUC-201806080302 OUC-201812050305 OUC-201812050306
Status Holotype Paratype Paratype Paratype Paratype Paratype Paratype
Sex ♂ ♂ ♂ ♂ ♂ ♀ ♀ ♀ ♀
Maturity mature mature mature mature mature immature immature mature mature
TL 212.7 216.3 212.1 258.6 283.9 195.7 208.6 234.8 382.1
TW(g) 30.6 24.5 37.3 28.8 23.4 17.6 16.1 25.1 39.2
ML 45.5 49.5 53.2 56.3 63.2 41.7 50.7 51.7 83.3
MWI 72.5 69.7 65.6 66.2 65.0 64.5 60.5 77.4 46.2
HWI 35.2 29.1 23.9 30.7 27.2 30.0 27.6 39.1 23.0
PAI 120.9 116.6 111.5 116.5 103.2 149.6 111.0 125.0 98.3
FLI 63.1 65.1 66.2 67.1 53.2 61.4 57.8 68.5 51.0
FFLI 46.0 35.1 23.9 26.2 25.9 40.6 32.4 34.2 33.6
WDI 17.0 17.3 22.9 15.7 18.3 19.8 20.0 16.9 16.2
AL1I 333.8 331.9 237.2 322.7 196.7 D 285.4 324.8 294.5
AL2I 336.3 276.0 241.2 302.1 304.1 322.5 251.1 313.5 154.9
AL3I 291.9 273.1 231.8 220.1 220.4 294.5 226.8 272.7 293.0
AL4I 296.7 259.2 193.2 265.2 229.9 299.0 211.8 281.8 237.1
AWI 15.2 13.8 14.3 13.2 11.6 14.5 18.0 16.2 8.7
LLI 7.0 11.6 9.8 9.6 10.4 – – – –
CaLI 31.6 26.3 28.2 30.9 31.1 – – – –
HAMI 184.2 160.8 162.4 182.8 155.5 – – – –
OAI 63.1 58.9 70.1 81.8 70.6 – – – –
SDI 6.9 6.5 5.7 6.6 6.5 5.4 5.0 5.9 5.4
HASC 79 82 83 83 70 – – – –
SC 157 191 171 198 195 191 163 178 177
GC 8 9 8 9 8 8 8 8 8
SpC – 6 8 – 4 – – – –
SpL – 37.8 58.1 – 79.1 – – – –
SpW – 1.4 1.5 – 1.6 – – – –
EgC – – – – – – – 64 67
EgL – – – – – – – 14.0 14.5
EgW – – – – – – – 4.3 3.3
UHL/UCL – 0.31 0.28 0.28 0.29 – – 0.32 0.28
URW/UCL – 0.18 0.13 0.17 0.15 – – 0.16 0.12
URL/UHL – 0.33 0.28 0.38 0.23 – – 0.27 0.23
LHL/LCL – 0.38 0.39 0.40 0.45 – – 0.34 0.36
LRW/LCL – 0.33 0.32 0.41 0.36 – – 0.26 0.30

Digestive tract (Fig. 3A). Anterior salivary glands small, approximately one-third length of buccal mass. Posterior salivary glands triangular and smaller than buccal mass. Oesophagus long. Spiral caecum with one whorl. Intestine long. Digestive gland well developed, brown. Ink sac present, embedded in the digestive gland and attached to the intestine posteriorly. Ink sac opening into the anus. Anal flaps small.

Figure 3. 

Callistoctopus xiaohongxu sp. nov. A digestive system, female, 83.3 mm ML (OUC-201812050306) B upper beak, lateral view, female, 50.7 mm ML (OUC-201806080302) C lower beak, lateral view, female, 50.7 mm ML (OUC-201806080302) D lower beak, ventral view, female, 50.7 mm ML (OUC-201806080302) E, F scanning electron micrograph of radulae, male, 53.2 mm ML (OUC-201812050302). Abbreviations: a, anus; asg, anterior salivary gland; bm, buccal mass; c, crest; ca, caecum; cr, crop; dg, digestive gland; h, hood; i, intestine; is, ink sac; lw, lateral wing; o, oesophagus; psg, posterior salivary gland; r, rostrum; st, stomach; w, wing. Scale bars: 10 mm (A–D); 200 μm (E, F).

Upper beak (Fig. 3B) with short rostrum, narrow hood, and slightly curved crest. Ratios of upper beak measurements 0.28–0.32 for UHL/UCL, 0.12–0.18 for URW/UCL, and 0.23–0.38 for URL/UHL. Lower beak (Fig. 3C, D) with a blunt rostrum, narrow hood, moderately broad wings and flared lateral walls separated in posterior, posterior notch deep. Radula (Fig. 3E, F) with 7 teeth and 2 marginal plates per transverse row. Ratios of lower beak measurements 0.34–0.45 for LHL/LCL and 0.26–0.41 for LRW/LCL. Rhachidian tooth with 1–2 lateral cusps on each side; first lateral teeth small, sharp; second lateral teeth broad-based triangular, larger than first, sharp; marginal teeth long, curved, sharply pointed, longer than second lateral teeth; marginal plates flat.

Male reproductive tract (Fig. 4A). In mature males, the terminal organ inverse 6-shaped. Spermatophore storage sac long. Accessory gland curved, longer than spermatophore storage sac. Spermatophore gland long. Vas deferens very short, narrow. Testis roundish, small. Spermatophores (Fig. 4B) of moderate size, average length 60 mm, approximately 75% ML, narrow (average 1.5 mm in width); approximately 4–8 spermatophores in storage sac.

Figure 4. 

Callistoctopus xiaohongxu sp. nov A reproductive system of male, 56.3 mm ML (OUC-201812050303) B spermatophore, male, 56.3 mm ML (OUC-201812050303) mm C reproductive system of female, 51.7 mm ML (OUC-201812050305) D egg cluster, female, 51.7 mm ML (OUC-201812050305) E single egg female, 51.7 mm ML (OUC-201812050305). Abbreviations: ag, accessory gland; cb, cement body; do, distal oviduct; ea, ejaculatory apparatus; o, ovary; og, oviducal gland; sg, spermatophore gland; sr, sperm reservoir; ss, spermatophore storage sac; t, testis; tf, terminal filament; to, terminal organ; vd, vas deferens. Scale bars: 10 mm.

Female reproductive tract (Fig. 4C–E). Ovary large, round in mature females. Two distal oviducts long. Two oviducal glands wider than distal oviducts. Mature females with approximately 65 large eggs (average 14.3 mm in length).

Integument (Fig. 5A). Colour of live animal reddish-orange. Animal turning white when stressed or post mortem. In live animals, a linear structure appears on the tissue connecting two adjacent arms, forming a net-like structure (Fig. 5B). Arm chromatophores under the skin distinct.

Figure 5. 

Callistoctopus xiaohongxu sp. nov. A live specimen B net-like structure on web.

Etymology

The name ‘xiaohongxu’, which refers to its small body size and reddish body colour, is the phonetic translation of the local Chinese name of this species in Zhangzhou, where specimens were collected.

Distribution

According to fishermen in Zhangzhou, this species is distributed in the East China Sea and the South China Sea, mainly in Quanzhou, Fujian Province to Shanwei, Guangdong Province.

Molecular analyses

Phylogenetic analyses were performed based on the fragments of the COI gene using Maximum likelihood (ML) and Bayesian inference (BI) methods. Fragments 593 bp in length were obtained from the mitochondrial COI gene of nine specimens. Both ML and BI trees showed a similar topology (Fig. 6) with a highly supported monophyletic clade (bootstrap value [BS] = 94%, posterior probability [PP] = 100%) containing all nine specimens identified as Callistoctopus xiaohongxu sp. nov. C. xiaohongxu sp. nov. belonged to the clade of ‘O’. minor and other four species of Callistoctopus with [BS] = 82% and [PP] = 89%, respectively. Moreover, the COI gene analyses suggested that species of the genus Octopus used in this study were not clustered into one clade. Additionally, the genetic distance of C. xiaohongxu sp. nov. and the other 16 Octopodidae species ranged from 11.13 to 21.09% (Table 3).

Table 3.
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Pairwise comparison of the genetic distances among Octopodidae species based on the COI gene. Abbreviations: A. a., Amphioctopus aegina; A. f., Amphioctopus fangsiao; A. n., Amphioctopus neglectus; A. r., Amphioctopus rex; Ca. a., Callistoctopus aspilosomatis; Ca. l., Callistoctopus luteus; Ca. m., Callistoctopus macropus, Ca. o., Callistoctopus ornatus; Ca. x., Callistoctopus xiaohongxu; Ci. c., Cistopus chinensis; Ci. t., Cistopus taiwanicus; O. b., Octopus bimaculatus; O. co., Octopus conispadiceus; O. cy., Octopus cyanea; ‘O.’ m., ‘Octopus’ minor; O. s., Octopus sinensis; and O. v., Octopus vulgaris.

A. a. A. f. A. n. A. r. Ca. a. Ca. l. Ca. m. Ca. o. Ca. x. Ci. c. Ci. t. O. b. O. co. O. cy. ‘O.’ m. O. s.
A. a. –
A. f. 14.29 –
A. n. 12.43 16.36 –
A. r. 11.78 16.56 11.03 –
Ca. a. 20.32 20.34 22.76 21.09 –
Ca. l. 20.10 19.02 20.18 19.50 14.54 –
Ca. m. 19.62 20.57 21.27 20.37 12.22 15.12 –
Ca. o. 21.03 20.38 19.63 21.13 10.95 15.40 5.01 –
Ca. x. 18.51 19.40 20.29 18.63 14.04 15.54 12.18 11.97 –
Ci. c. 18.26 18.81 20.97 19.47 21.40 20.55 19.70 20.22 17.76 –
Ci. t. 16.45 19.01 16.93 18.05 19.87 21.11 18.49 17.58 19.08 13.11 –
O. b. 18.05 14.74 20.21 20.68 20.40 19.27 19.96 20.50 17.40 16.28 19.04 –
O. co. 20.09 19.22 18.73 20.36 16.94 17.68 19.02 17.39 16.63 20.87 20.34 21.17 –
O. cy. 14.95 16.74 17.47 18.61 17.19 17.65 17.83 17.16 15.33 17.17 16.24 15.00 18.05 –
‘O’. m 19.87 19.44 23.52 22.04 13.50 14.17 10.77 11.43 11.13 18.28 20.60 19.21 19.01 17.59 –
O. s. 14.30 17.90 17.47 18.37 21.07 20.44 20.35 20.36 20.72 17.68 17.93 15.27 20.36 16.99 20.81 –
O. v. 14.37 17.51 17.23 18.29 21.51 21.04 20.14 20.15 21.09 18.87 18.60 15.21 20.69 18.30 20.69 2.97
Figure 6. 

Phylogenetic trees derived from Maximum likelihood (ML) and Bayesian inference (BI) methods based on the COI gene. Numbers at each node are bootstrap (left) for ML and posterior probability (right) for BI analyses, respectively.

Discussion

As mentioned previously, Callistoctopus xiaohongxu sp. nov. has been mistakenly identified and sold in fish markets as the juveniles of ‘O’. minor, because they are similar in having smooth skin and reddish-brown colour in chilled specimens. However, C. xiaohongxu sp. nov. and ‘O’. minor can be readily distinguished by the morphological characteristics compared in Table 4. Callistoctopus xiaohongxu sp. nov. has:

Table 4.
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Comparison of key morphological characters between Callistoctopus xiaohongxu sp. nov., Callistoctopus species, and ‘Octopus’ minor Sasaki, 1920.

Item Callistoctopus xiaohongxu sp. nov. Callistoctopus species ‘Octopus’ minor Sasaki, 1920
Data source this study Norman et al. 2014 Norman et al. 2014
Colour reddish-orange to reddish-brown, no spot Typically, red-brown to red, white spots or bars on mantle, head and arms red-brown, light yellow spots on mantle surface
Sculpture smooth smooth or with scattered low papillae smooth
GC 8–9 10–14 10–12
Funnel organ \ /\ /-shaped W, UU or VV-shaped V V-shaped
WDI 15.7 to 22.9 around 7 to 28 deepest around 10
ALI 154.9–336.3 300–800 400–500
OAI 58.9–81.8 around 40–95 around 50
Enlarged suckers absent absent present
Ligula cylindrical with groove, LLI 7.0–11.6 cylindrical with deep groove, LLI around 1.5–9 spoon-like with wide hollow groove, LLI around 18–23
  1. no spots on mantle surfaces (‘ O’. minor has light yellow spots);
  2. a \∧/-shaped funnel organ (the funnel organ shape of ‘ O’. minor is V V-shaped);
  3. gills with 8 or 9 lamellae per demibranch (10–12 lamellae per demibranch in ‘ O’. minor);
  4. no enlarged suckers in mature males (‘ O’. minor has enlarged suckers);
  5. cylindrical ligula with groove (spoon-like with a wide hollow groove in ‘ O’. minor).

Callistoctopus xiaohongxu sp. nov. is also distinct from other species of Callistoctopus (Table 4). Compared to the key morphological characters, C. xiaohongxu sp. nov. has no spot on the skin (vs other Callistoctopus species that have white spots or bars on the mantle, head, and arms), fewer gill lamellae (gill lamellae 8–9 vs 10–14 in Callistoctopus species), funnel organ \ /\ /-shaped (vs W, UU or VV-shaped in other species), and relatively shorter arms (ALI 154.9–336.3 vs 300–800 in the other Callistoctopus species).

Judging from the K2P genetic distance (Table 3), C. xiaohongxu sp. nov. can be separated from the other 16 species of Octopodidae by genetic distances ranging from 11.13 to 21.09%. According to the phylogenetic tree (Fig. 6), C. xiaohongxu sp. nov. has a close relationship to ‘O’. minor and four species of Callistoctopus with [BS] = 82% and [PP] = 89%. However, the taxonomic status of ‘O’. minor is unresolved, and it is placed in the genus Octopus provisionally. Besides, the very limited suite of molecular data suggested that genetic relationships among species of the genus Callistoctopus need further studies. Still, for the accurate phylogenetic status of ‘O’. minor, more research would be required to establish the relationships among species of Octopus and Callistoctopus.

Octopus is one of the most species-rich cephalopod genera but was considered a ‘catch-all’ genus by Guzik et al. (2005). It is not monophyletic in its current composition and needs revision and robust phylogenetic analyses (Strugnell et al. 2005; Dai et al. 2012; Amor et al. 2015; Ritschard et al. 2019; Tang et al. 2020). In our study, species in Octopus were not clustered into one clade. Accordingly, our study supports the polyphyly of the genus Octopus.

We are planning to analyse the mitochondrial genome of C. xiaohongxu sp. nov. in the future. Better taxon sampling would facilitate a better understanding of octopod phylogeny as well as a better substantiated generic assignment of C. xiaohongxu sp. nov.

Acknowledgements

We would like to thank Dr Hong Zhong, Ms Feige Sunxie for collecting specimens, and Poungthong Itsaret for helping to take a few photos of the new species. We are also grateful to Prof ChungCheng Lu for the valuable advice for improving the manuscript. This study was supported by the National Natural Science Foundation of China (No.31672257, 32170536).

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