The Seven-arm Octopus , Haliphron atlanticus Streenstrup , 1861 ( Cephalopoda , Alloposidae ) , in the Fernando de Noronha archipelago , Brazil

The rare deep-sea octopus, Haliphron atlanticus is the only known species recognized within the genus. A fragment of H. atlanticus was found in the Fernando de Noronha archipelago, Brazil (South Atlantic). Both phylogenetic reconstruction and pairwise genetic divergence show that the specimen recorded in South Atlantic is closely related to individuals from North Pacific. However, there is a greater divergence among these specimens and a giant octopus from North Atlantic. This evidence suggests that Haliphron is not monospecific, with at least two species, both represented in the Atlantic Ocean.

The rare gelatinous Seven-arm Octopus, Haliphron atlanticus Streenstrup, 1861 is the only known species recognized within the genus Haliphron Steenstrup, 1861 (Alloposidae).This giant octopus is a benthopelagic species with a circumglobal distribution, reaching a total length up to 4 m (Alvariño & Hunter 1981;Young 1995;Finn 2014).It lives in a depth range from about 1200 m, over depths up to 6500 m (O'Shea 2004;Finn 2014).It is believed that this species lives in pelagic habitats during certain phases of its life cycle (mainly juveniles), and inhabits the seabed of the continental slope during other phases (Thore 1949;O'Shea 2004).
Notwithstanding, records of this species are rare and only few adults are known (O'Shea 2002).Furthermore, most specimens are incomplete, sometimes restricted to a single arm fragment, as the type material, limiting systematics accounts (O'Shea 2004).On the other hand, according to Alvariño & Hunter (1981), H. atlanticus may not be as rare as it seems, and, mainly adults, are probably adept at avoiding nets of all types.
The most remarkable morphological characters are the gelatinous and well-pigmented body; deep web between all arms; and a single series of suckers near the mouth, which become biserial closer to edge of the web.Males are smaller than females and have the third right arm hectocotylized, detachable, and developed in pouch in front of the eye; this gives the impression that it has only seven arms (Thore 1949;Willassen 1986;Young 1995;O'Shea 2004;Finn 2014).
Given the difficulty of obtaining data on deep-sea cephalopods, and consequently, the lack of information in the literature about this group, this study validates the occurrence of H. atlanticus in Southwest Atlantic.In addition, it aims to provide molecular data for future phylogenetic and phylogeographic studies involving this species.
A fresh fragment of H. atlanticus (arms and part of the mantle) was found in November 2015 floating in the water column to a depth of 4 m, near Rata Island, in the Fernando de Noronha Marine National Park.Rata Island is part of the Fernando de Noronha archipelago, in the Tropical Southwest Atlantic province, about 360 km off the northeastern coast of Brazil (03°48ʹ53.05ʺS, 032°24ʹ14.13ʺW) (Figure 1).The fragment was collected by hand (permit SISBIO 10706-5) and tissue samples were stored at 95% ethanol.Part of the animal was frozen and the tissue samples were deposited in the Collection of Invertebrates at the Universidade Federal do Rio Grande do Norte (CTR 4560).
Cytochrome oxidase subunit I gene (COI) amplicons were obtained using universal primers LCO and HCO  (Folmer et al. 1994).The PCR amplification reaction was conducted in a final volume of 25 µL containing 1 µL forward primer (10 mM), 1 µL reverse primer, 12.5 µL Taq DNA Polymerase Master Mix Ampliqon, 8.5 µL H 2 O and 2 µL DNA.PCR cycle parameters used for COI were 3 min at 95°C for denaturation, followed by 40 cycles of 1 min at 94°C, 1 min at 45°C for annealing, 1.5 min at 72°C for extension and a final extension step of 4 min at 72°C.Finally, 25 µL of the PCR products were purified and sequenced by Macrogen Inc, Seoul, Korea.
The electropherograms were edited in Geneious 9.0.2 (http://www.geneious.com,Kearse et al. 2012) and sequences were aligned using MEGA 6 (Tamura et al. 2013).A molecular phylogenetic analysis (Bayesian Coalescent reconstruction) was carried out in BEAST 1.75 (Drummond et al. 2012).The substitution model GTR+G was used for COI, as suggested by jModeltest (Posada & Crandall 1998).An estimated rate of evolution for cephalopods (3.81 substitutions per site per billion years) was used in the Bayesian analyses (Strugnell et al. 2012).A total of 5 × 10 7 MCMC runs, saving of one every 5×10³ runs was performed, 10,000 trees were saved and the first 15% removed as burnin.A consensus tree accessing the posteriori probabil-ity values of each clade was generated using TreeAnnotator 1.6.1 (Drummond et al. 2012).
A pairwise matrix of Kimura 2-parameter (K2P) was also performed in MEGA 6 (Tamura et al. 2013) to compare the genetic distances within and among other octopus species.COI sequences of some incirrate (cirrus absent) octopuses available in GenBank were also used in this study (Table 1).
The specimen was incomplete (Figure 2A) and the correct identification was only possible after molecular analysis (Table 1).However, some diagnostic features related to the gelatinous long arms and biserial arrangement of the distal suckers were observed (Figure 2B).Molecular analysis using 615 bp fragments of COI mitochondrial gene of 14 individuals belonging to 11 species (pelagic and benthonic octopods) corroborated the morphological identification (Table 1).The phylogenetic reconstruction showed that all sequences identified as H. atlanticus form a monophyletic and well-supported clade (Posterior Probability -PP = 1) (Figure 3).The specimens from North Pacific are more closely related to the Brazilian specimen than the other individual from the Atlantic Ocean (USA), which is the sister group of this clade.According to phylogenetic analysis, the sister species of H. atlanticus is the deep-sea Tremoctopus    violaceus Delle Chiaje, 1830, a widespread pelagic species found in the Atlantic Ocean (PP = 0.93).Carlini et al. (2001) and Strugnell et al. (2005) also found a close relationship between these species, using morphological evidence and multi-gene approach, respectively.Both phylogenetic reconstruction and pairwise genetic divergence show that the specimen recorded in South Atlantic is closely related to individuals from North Pacific, Hawaii (K2P = 0.005), and Canada (K2P = 0.003) (Table 2).However, there is a greater divergence, between 2.9%-3.1%,among these specimens and the giant octopus of North Atlantic, which is about twice the distance among congeneric species, 1.5% between Pareledone charcoti Joubin, 1905 and P. albimaculata Allcock, 2005.The specimen from Fernando de Noronha archipelago is the first reliable record of the genus Haliphron in Southwest Atlantic.To date, H. atlanticus was only recorded in Brazilian waters through beaks identified from stomach contents of long-distance foraging marine predators, as sharks and tunas (Santos & Haimovici 2002;Vaske-Júnior 2005).Apparently, the fragment was not regurgitated by large predators, because it was large, fresh, and well pigmented.This fragment also did not drift from a distant area by oceanic currents, because it would have quickly been eaten by pelagic migrants, such as sharks, tunas and whales, or seabirds such as albatrosses.We believe that part of the giant octopus was predated around the Fernando de Noronha archipelago and the remaining fragment drifted in the water column until arriving in shallow waters.
Phylogenetic reconstruction confirmed the taxonomic identity of the specimen fragment within the monotypic Haliphron clade.Furthermore, the analysis of pairwise genetic divergence showed intraspecific variation lower than 1% (except the North Atlantic specimen), which is consistent with barcoding proposals in many coleoid cephalopods (Dai et al. 2012).However, the K2P distances among H. atlanticus from North Atlantic and the others specimens were between 2.7% to 3.1%, similar to interspecific variation between octopod congeners (Kaneko et al. 2011).In a molecular identification study of 54 octopods species, Undheim et al. (2010) found that variation between congeners was in most cases higher than 2%, but ranged from 1.0% to 7.7%.Allcock et al. (2011) also verified small interspecific variation in COI sequences of the Pareledone genus (mean 2.2% K2P).These evidence suggest that Haliphron is not monospecific with, at least, two species, both represented in the Atlantic Ocean.O'Shea (2004) had also suggested an additional species of Haliphron based on morphological differences among specimens from New Zealand and Hawaii.Further morphological and molecular systematics studies using more specimens are needed to confirm this hypothesis.
The giant H. atlanticus is usually, but not exclusively, associated with islands or continental slopes (O'Shea 2004).According to some studies, H. atlanticus might pass periods of its life cycle alternating between the open waters and sea bottom (Thore 1949;Alvariño & Hunter 1981;Willassen 1986;O'Shea 2004).Furthermore, some captures with nets suggest that H. atlanticus may undertake diel vertical migration (Finn 2014).Thus, the oceanic islands, an oasis on the open sea, can provide a productive environment for feeding and/or spawning activity for this species.
The new record of H. atlanticus in Fernando de Noronha archipelago will contribute for global systematic studies about this rarely encountered giant octopus.In addition, it should be pointed out the importance of oceanic islands to the deep-sea fauna, reinforcing the need to protect these unique environments for the maintenance of marine biodiversity.

Figure 1 .
Figure 1.Geographical location of the Fernando de Noronha archipelago, where Haliphron atlanticus was recorded.The archipelago in detail is shown above, with white circle highlighting the sampling area of the giant octopus.In the global map, the dark circles indicate records of adults around the world.The open circles are the locations of samples used in the molecular analysis.

Figure 2 .
Figure 2. Collection of Haliphron atlanticus gelatinous body fragment (A) and detail of the biserial arrangement of distal suckers in the giant octopus arms (B).Photos: Liana Mendes (A) and Leonardo Veras (B).

Figure 3 .
Figure 3. Molecular phylogeny constructed using Bayesian inference for a partial fragment of the Cytochrome Oxidase I gene.The Bayesian posterior probabilities are shown below nodes.The specimen collected in this study is highlighted with an asterisk.

Table 1 .
List of octopus specimens used in this study showing the sampling regions, accession number of DNA sequences, geographical coordinates and references.All sequences are obtained from GenBank, except the sequence with an asterisk (downloaded from BOLD database).