Morphological description and mitochondrial DNA-based phylogenetic placement of a new species of Callistoctopus Taki, 1964 (Cephalopoda, Octopodidae) from the southeast waters of China

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


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.

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.
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).
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.
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.

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  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. 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. spoon-like with wide hollow groove, LLI around 18-23