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Phylogeographical features of octopus vulgaris and octopus insularis in the Southeastern Atlantic based on the analysis of mitochondrial markers.

ABSTRACT The genus Octopus occurs in tropical and temperate oceanic waters throughout the world, and currently includes 112 species, although the phylogenetic relationships among the different taxa are still poorly understood. The cosmopolitan Octopus vulgaris is one of the most widely analyzed cephalopods in genetic studies, primarily because of its ample range and the problems associated with the morphological identification of specimens, which indicate the possible existence of a species complex with a worldwide distribution. Two large-bodied octopus species--O, vulgaris and Octopus insularis--are found in the western South Atlantic. The limits of the geographical range of the O. insularis are still unclear. The current study is based on a phylogeographic analysis of the 2 species in the South Atlantic, with the objective of confirming their monophyletic status and the limits of their geographical distribution in this region. The analyses were based on the mitochondrial genes 16S rDNA and Cytochrome Oxidase subunit I (COI). The topologies generated for both genes confirmed the monophyletic status of the 2 species. In the case of O. vulgaris, it was possible to confirm the monophyletic status of the specimens from this region relative to those of other areas around the world, although 3 distinct haplogroups were clearly differentiated, corresponding to the Americas, Europe and Africa, and Asia. The differentiation among these 3 groups may be determined by the limitations of the dispersal of paralarvae among continents. Further studies are needed to confirm the possible occurrence of distinct groups in the western South Atlantic, as well as the influence of oceanic currents on the phylogeographical distribution of O. vulgaris on the Brazilian coast.

KEY WORDS: phylogeography, Octopus vulgaris, Octopus insularis, South Atlantic, genetics, mitochondrial DNA

INTRODUCTION

The genus Octopus occurs throughout the tropical and temperate regions of the world's oceans (Norman 2003). Approximately 112 species are currently recognized, although the phylogenetic relationships among the different forms are still poorly understood (Norman & Hochberg 2005). The nominal members of this genus present widely varying characteristics, ranging from small-bodied species with large eggs, low fecundity, benthic larvae, and a restricted geographical distribution, such as Octopus tehuelchus Orbigny, 1834 (Alves& Haimovici 2011), to large, widely distributed species with high fecundity and pelagic postlarvae, such as Octopus vulgaris Cuvier, 1797 (Mangold 1987, Villanueva & Norman 2008). However, recent phylogenetic studies have indicated that O. tehuelchus, in fact, is related phylogenetically to Grimpella and Callistoctopus, not Octopus (Acosta-Jofre et al. 2012).

Genetically, Octopus vulgaris is one of the most widely studied cephalopod species (Carlini & Graves 1999, Warnke 1999, Warnke et al. 2004, Guzik et al. 2005, Leite et al. 2008), which is a result of a combination of its cosmopolitan distribution and the difficulties of identifying the species based on morphological criteria. Norman (2003) referred to this taxon as a "species complex," and argued that a number of distinct taxa are classified incorrectly as Octopus vulgaris in different parts of the world. This has been confirmed in recent years by a number of genetic and morphological studies, principally in the western hemisphere, which resulted in the description of a number of new species, including Octopus maya (Voss & Ramirez 1966), Octopus mimus (Guerra et al. 1999), and Octopus insularis (Leite et al. 2008). The cosmopolitan distribution of O. vulgaris has been challenged by some authors (e.g., Mangold 1997, 1998), although its occurrence has been confirmed by the molecular genetic analysis of specimens from coastal waters of the Americas (Warnke et al. 2004, Sales et al. 2007), Africa (Oosthuizen et al. 2004), and Asia (Takumiya et al. 2005).

At least 2 species of large-bodied octopi with small eggs, high fecundity, and pelagic postlarvae occur in the western South Atlantic: Octopus vulgaris (Cuvier 1797) and Octopus insularis (Leite & Haimovici 2008). The geographical range of O. insularis, which was described from specimens collected in the vicinity of the oceanic islands off the northeastern coast of Brazil, is now known to include northern South America (Sales et al. 2007).

In the current study, a phylogeographical analysis of these 2 common Octopus species from the South Atlantic (Octopus vulgaris and Octopus insularis) was conducted using molecular mitochondrial markers. This analysis aimed to corroborate the identification of the species and their monophyletic status, as well as confirm their occurrence throughout the study area.

MATERIAL AND METHODS

Samples

Samples of the 2 study species were obtained along the coast of the western Atlantic in Brazil, between the latitudes 03'24'27" N and 27[degrees]08'48.06" S (Fig. 1). The specimens collected in northern Brazil (Amapa and Para states) were obtained from the bycatch of fishing for red snapper (Lutjanus purpureus Poey 1875) and green lobster (Panulirus laevicauda Latreille 1817), as well as from the stomach contents of some red snapper specimens (samples OvuPA 78, OvuPA 173, Ovu 184, OvuAP 225, and AmspPA 86, representing Amphioctopus sp.). All other specimens were obtained from commercial fisheries (Octopus hummelincki Adam 1936, Eledone massyae Voss 1964) or local fish markets (locations provided in Appendix A (Strugnell et al. 2004, Allcock et al. 2006, Teske et al. 2007)). A small fragment of muscle tissue was extracted from 1 of the arms of each animal, and was stored in a freezer in flasks with 100% ethanol until the extraction of the DNA.

Adult specimens were identified based on the specific literature (Roper et al. 1984). Some of these adults, as well as all the material obtained from stomach contents, were fixed in 10% formalin and deposited in the zoological collection of the Oceanographic Museum at Universidade Federal do Rio Grande (FURG). The identification of some of the specimens obtained from stomach contents, which were in an advanced stage of decomposition, and thus lacked the morphological structures necessary for taxonomic analysis, was achieved by comparing the DNA 16S and cytochrome oxidase subunit I (COl) sequences with those available for the study species in GenBank.

Extraction of DNA, Polymerase Chain Reaction, and Sequencing

Total DNA was isolated using the modified phenol/chloroform protocol of Sambrook and Russel (2001). When this approach was unsuccessful, a Wizard Genomics DNA purification kit was used, according to the manufacturer's instructions (Promega Corporation, Madison, WI). In both cases, the tissue was prewashed with 600 [micro]L ultrapure water based on two 2-min centrifugations at 13,000g for the removal of excess alcohol.

The primers for the 2 mitochondrial genes (16S rDNA and Cytochrome Oxidase subunit I--COI) were obtained from the literature (Table 1). Amplification of the 16S gene was based on the following cycling parameters: 2 min at 94[degrees]C for denaturation, followed by 30 cycles of 30 sec at 94[degrees]C, 1 min at 51 [degrees]C for annealing, and 2 min at 72[degrees]C for extension, and then 7 min at 72[degrees]C for the final extension. For COI, the procedure was 2 min at 94[degrees]C for denaturation, followed by 30 cycles of 1 min at 94[degrees]C, 1 min at 45.5[degrees]C for annealing, 2 min at 72[degrees]C for extension, and 7 min at 72[degrees]C for the final extension. The polymerase chain reactions for both markers were conducted in a final volume of 25 [micro]L containing 4 [micro]L DNTPs (1.25 mM), 2.5 [micro]L buffer solution (10X), 1 [micro]L Mg[Cl.sub.2] solution (50 mM), 80-200 ng total DNA, 0.25 [micro]L each oligonucleotide (200 ng/[micro]L), 0.25 [micro]L AccuPrime Taq enzyme polymerase (Invitrogen; 5 U/[micro]L), and sterile bidistilled water to complete the final reaction volume.

Prior to sequencing, the polymerase chain reactions were purified with the ExoSAP-IT enzyme (Amersham Pharmacia Biotech Inc.). Sequencing was conducted using BigDye kit reagents (Applied Biosystems), with the products being read in an ABI 3500 automatic sequencer (Applied Biosystems). Additional sequences from other Octopus species (Octopus vulgaris, Octopus insularis, Octopus maya Voss & Solis 1966, Octopus mimus Gould 1852, and Octopus bimaculoides Pickford & McConnaughey 1949), as well as Hapalochlaena maculosa Hoper & Hochberg 1988, were obtained from GenBank for the comparative analysis of the divergence among sequences and the rooting of the phylogenetic groups (details are provided in Appendix A (Strugnell et al. 2004, Allcock et al. 2006, Teske et al. 2007)).

Phylogenetic and Population Inferences

The DNA sequences were aligned using the Clusta1W multiple alignment tool (Thompson et al. 1997) in the BioEdit program v.5.0.6 (Hall 1999). After automatic alignment, each sequence was inspected visually for the correction of possible edition errors. This was especially important in the case of the 16S gene, which presented a large number of gaps when comparing sequences of the most divergent species.

For the phylogenetic analyses, the optimum evolutionary models were selected using the jModelTest program (Guidon & Gascuel 2003), based on the Akaike information criterion (Akaike 1974) for maximum likelihood (ML) and the Bayesian information criterion for Bayesian inference (BI). The ML analysis was run in PhyML 3.0 (Guidon et al. 2010), with the reliability of the groups being verified using a nonparametric bootstrap analysis with 1,000 replicates (Felsenstein 1985). The Bayesian analysis was run in MrBayes v 3.1.2 (Ronquist & Huelsenbeck 2003). For BIs, the data set was analyzed with a single substitution model (i.e., unpartitioned), and partitioned by gene and codon position (i.e., a separate substitution model was chosen for each of the 3 COIs). Partitioned Bayesian analyses were based on the Markov chain Monte Carlo sampling procedure, with 4 simultaneous runs, each consisting of 4 chains (1 cold, 3 heated), and a total run length of 10 million generations, using the parameters of the evolutionary models selected for each partition. The a posteriori Bayesian probabilities were selected by the 50% consensus rule, with random starting trees and trees sampled every 5,000 generations after the removal of the trees that appeared to have reached a stationary state, at which the burn-in was verified by the empirical examination of the likelihood values. FigTree v.1.1.2 was used to edit the phylogenetic trees. When the topologies were obtained, the observed clades were considered to be distinct groups for the subsequent calculation of intra- and interspecific divergence values in MEGA 5.04 (Tamura et al. 2011).

For the analysis of Octopus vulgaris and Octopus insularis populations, the indices of haplotype (h) (Nei 1987) and nucleotide diversity ([pi]) (Nei 1987) were estimated in DnaSP, version 5.10 (Librado & Rozas 2009). Arlequin 3.01 (Excoffier et al. 2006) was used to estimate the fixation indices ([F.sub.st]) (Weir & Hill 2002) and to run the hierarchical analysis of molecular variance (AMOVA) (Excoffier et al. 1992), which was based on 1,000 permutations using the Kimura 2P substitution model (Kimura 1980). The D (Tajima 1989) and Fs (Fu 1997) tests of selective neutrality were run in Arlequin 3.01 (Excoffier et al. 2006). The spatial distribution of haplotypes within the populations was mapped using Haploview (Salzburger et al. 2011).

RESULTS

A total of 948 bp were sequenced, including 482 for COI and 466 for 16S. The optimum models of substitution selected by jModelTest were TIM3 + G for the 16S gene (for both ML and BI), whereas for COI, different models were selected for ML (GTR + G) and BI (TIM2 + G) for the unpartitioned data set, and TIM 2 + I + G for the codon partitioned data set. Because the topologies produced by the 2 approaches were highly similar, only the ML trees are shown here (Figs. 2 and 3). The monophyletic status of both Octopus vulgaris and Octopus insularis is clear from the configuration of this tree.

The analysis indicated the presence of a single monophyletic Octopus vulgaris clade throughout the study area, with strong statistical support (99% for both ML and BI). Three distinct haplogroups can be discerned in the tree for the 16S gene in both phylogenetic approaches (Fig. 2). Group 1 is formed by specimens from Africa and Europe, whereas group 2 is formed exclusively by specimens from the southeastern Atlantic, including individuals from Venezuela and the coast of Brazil. Group 3 is composed of specimens from Asia (Japan and Taiwan), and is the most basal within the O. vulgaris clade. The topologies derived from the analysis of the COI gene also confirmed the monophyletic status of this species (statistical support, 99/1), as well as the presence of subgroups, although with a slightly different topology.

Nucleotide divergence between the different Octopus vulgaris groups ranged from 1.6-2.1% (Table 2). In turn, O. vulgaris diverged from other Octopus species by 7.8-9.7% (Octopus bimaculoides), 9.5-11.2% (Octopus insularis), 11.7-12.9% (Octopus mimus), and 13.2-13.6% (Octopus hummelincki). The lowest genetic divergence between 2 species was 5.2% for O. insularis and O. mimus. Genetic divergence among genera ranged from 10-26%.

The species Octopus insularis was also clearly monophyletic (77/1) based on the 16S sequences, but closely related phylogenetically to Octopus mimus from the Pacific Ocean, as indicated by the low genetic divergence recorded between the species (Fig. 2, Table 2). The CO1 sequences also confirm the monophyletic status of this species. Because CO1 sequences were not available for Octopus mimus, Octopus maya was the closest species to O. insularis in this phylogenetic analysis, followed by Octopus bimaculoides, Octopus vulgaris, and Octopus hummelincki (Fig. 3, Table 3).

Based on the identification of the 3 subgroups in Octopus vulgaris, 3 geographical divisions were established (Appendices B and C): group 1, specimens from the western hemisphere: group 2, specimens from Europe and the eastern Atlantic; and group 3, specimens from Asia. The databases for the 16S and CO1 genes include some unique samples, of which the number varies according to the number of taxa included in the analysis (63 in 16S and only 46 in COI). In the case of the 16S gene, the O. vulgaris subgroups presented high values for both genetic and haplotype diversity, ranging from 0.81-1.00 (Table 4). Group 1 presented the largest number of polymorphic sites, followed by groups 2 and 3. However, the highest haplotype diversity was recorded in the Asian group (group 3), the lowest in the African group (group 2), and none of the haplotypes were shared by the different populations.

Nucleotide diversity varied from 0.005 (for groups 1 and 2)-0.007 (for group 3). The haplotype networks generated from the sequences upheld the 3 subgroups, corresponding to their geographical distribution (Fig. 4).

This was confirmed by the high values obtained for the AMOVA and [F.sub.st] analyses, which indicate more divergence between than within populations (Table 5). In addition, all the between-population values for [PHI]st were significant (P < 0.05), with the greatest differentiation found between the populations of groups 1 and 3 (Table 6). The [PHI]st values obtained for 16S also presented some differences in comparison with those for COI. Although all the values for COI were highly significant (P < 0.05), the highest divergence was obtained for groups 1 and 2, and the lowest between groups 2 and 3. This gene also returned highly significant AMOVA and [F.sub.st] values for the Octopus vulgaris groups (Table 7). The distribution of polymorphic sites was also distinct in comparison with 16S. Group 3 presented 20 polymorphic sites, even though only 4 specimens were sequences, whereas the African group, despite being represented by 17 specimens, had the lowest number of polymorphic sites (Table 7). The CO1 gene also showed highly significant AMOVA and [F.sub.st] values for the O. vulgaris groups (Table 8). The [PHI]st values obtained for the 16S also presented some differences in comparison with those for COI. While all the values for COI were highly significant (P < 0.05), the highest divergence was obtained for groups 1 and 2, and the lowest between groups 2 and 3 (Table 9). The [PHI]st values for COI also presented certain differences in comparison with 16S. All the values were highly significant (P < 0.05), with the highest value being recorded between groups 1 and 2 (Table 9). In contrast with the 16S gene, however, a number of haplotypes were shared between groups 2 and 3. It is also interesting to note that 1 specimen from group 1 (OvuPA 173-H_3) was closely related to group 2 (Fig. 5), as observed in the phylogenetic tree generated for this gene (Fig. 3).

DISCUSSION

Octopus vulgaris

The phylogenetic analyses presented here confirmed the monophyletic status of Octopus vulgaris, with 3 well-defined continental groups. It is important to note that even though these groups are well defined and structured, the level of divergence among them is lower than that found typically between closely related species. The monophyletic status of the samples from the western hemisphere is especially important here, given that the largest number of specimens were obtained from this region. The existence of well-supported clades within the species indicates that each geographical region may support its own distinct O. vulgaris lineage. The occurrence of this species in the southeastern Indian Ocean was also confirmed recently, based on molecular markers and morphometric analyses, although some parameters were distinct from those presented by European specimens, such as a narrower head, smaller funnel, and larger number of suckers on the hectocotylus (Guerra et al. 2010).

Differentiation at the population level in cephalopods and, on a more ample temporal scale--speciation--may be derived from genetic, anatomic, physiological, or behavioral incompatibilities, reflecting the dispersal capacity of the planktonic larvae and/or the migratory potential of the adults (O'Dor 1988). The dispersal capacity of the juveniles depends on their size at the time of hatching and during the planktonic phase. The larger the juveniles, the shorter the planktonic phase, and the faster the transition to the adult lifestyle, when dispersal capacity is reduced (Boletzky 1987, Vecchione 1987).

Oceanic currents may limit the dispersal potential of the Octopus vulgaris paralarvae, restricting their migration among different regions. Previous studies of this species found little evidence of geographical differentiation or genetic distance among populations, nor of possible morphological differentiation consistent with the existence of distinct populations of O. vulgaris in different geographical regions. However, Vidal et al. (2010) recently found marked differences in the distribution of chromatophores in O. vulgaris paralarvae from the northeastern (Galicia, Spain) and southwestern Atlantic (southern Brazil), which reinforce the findings of the current study. These authors suggested the possible existence of distinct geographical populations of the species, or a cryptic species similar to O. vulgaris, reinforcing the need for the analysis of genetic divergence levels. In the current study, specimens from the same areas--Spain and Santa Catarina, in Brazil do not diverge genetically to a degree consistent with species-level differentiation. However, 1 specimen from Para, in northern Brazil (OvuPA 184, Fig. 3) was quite distinct phylogenetically from the other samples from the southwestern Atlantic, which indicates the possible presence of a cryptic species in the South American O. vulgaris species complex.

Murphy et al. (2002) analyzed microsatellites in Octopus vulgaris populations from the northwestern coast of Africa and found highly significant genetic structuring among specimens from Mauritania and the western Sahara. In a second microsatellite study, Cabranes et al. (2007) compared populations from the eastern Atlantic and the Mediterranean, and found a general trend for increasing genetic differentiation with increasing geographical distance, although the tendency was not upheld at distances of less than 200 km. Moreira et al. (2011) identified 4 subpopulations of O. vulgaris off southern Brazil, once again, with a tendency for greater genetic differentiation between geographically more distant populations.

The haplotype network for the COI gene also identified a close relationship between 1 individual from group 1 (OvuPA 173) and the members of the European and Asian groups. Initial evidence of intercontinental genetic similarity among a number of Octopus species was recorded by Warnke et al. (2004), who analyzed many of the specimens of Octopus vulgaris included in the current study (from all 3 groups), and also confirmed the monophyletic status of the species, with well-supported differentiation among continents (bootstrap values of 70-100), which is consistent with the results of the current study.

Octopus insularis

In a phylogenetic comparison between Octopus vulgaris from Europe and Octopus mimus from Central and South America, Soller et al. (2000) found that some specimens from the northern South Atlantic were genetically distinct from both species. These specimens were then formally described as a new species, Octopus insularis (Leite et al. 2008). The geographical range of this species was originally thought to be restricted to the oceanic islands off northeastern Brazil, although Sales et al. (2007) had collected specimens from the northern extreme of the South Atlantic. The results of the current study indicate that the species is distributed throughout the northern coast of Brazil, ranging as far south as Bahia, on the east coast.

This study also confirms the monophyly of the species as well as its affinities with some sympatric Octopus species. The range of this species is influenced by a number of different oceanic (the South Equatorial Current and the Equatorial Countercurrent) and continental (northern Brazilian and Brazilian) currents, which may favor the dispersal of the pelagic paralarvae toward both the open sea and coastal areas (Scheltema 1986, Lumpkin & Garzoli 2005). Based on the 16S gene, Octopus mimus was the Octopus species most closely related to Octopus insularis (with the lowest divergence for any 2 representatives of the genus), although in the COI topology (which did not include O. mimus), Octopus maya is the sister species of O. insularis. The low levels of genetic divergence observed here indicate that either O. maya or O. mimus may have shared the most recent common ancestor with O. insularis.

The genetic structuring found in both Octopus vulgaris and Octopus insularis, together with the pattern reported for other Octopus species (Murphy et al. 2002, Cabranes et al. 2007, Doubleday et al. 2009, Moreira et al. 2011), indicate that the association between genetic and geographical distances is a common feature of this genus. Specific factors such as direct internal fertilization (Kayes 1974, Mather 1988), a solitary lifestyle, and the reduced dispersal capacity of the adults (Hanlon & Messenger 1996) may combine to favor the genetic structuring of the populations of these animals.

The current study amplifies the geographical distribution of Octopus insularis along the Atlantic coast of South America, and confirms the monophyletic status of Octopus vulgaris throughout its worldwide range. The findings also generate an important question: Are the genetic differences among the O. vulgaris lineages consistent with species-level differentiation? The levels of nucleotide divergence found here (>1% for 16S and ~3% for COI) can certainly be considered evidence of supporting a taxonomic revision of this species, although this is a complex question that requires a more detailed analysis of a much wider samples of populations representing the different geographical lineages.

APPENDIX A
List of specimens analyzed in the current study, showing their
geographical origin, source, markers analyzed, and code numbers.

     Species                    16S

Octopus insularis                l
O. insularis                     l
O. insularis                     1
O. insularis                     1
O. insularis                     2
O. insularis                     --
O. insularis                     1
O. insularis                     --
O. insularis                     --
O. insularis                     --
O. insularis                     --
O. insularis                     --

O. insularis                     --
O. insularis                     1
O. insularis                     --

O. insularis                     --
O. insularis                     --
O. insularis                     --
O. insularis                     --
O. insularis                     --
O. insularis                     --
O. insularis                     --
O. insularis                     --
O. insularis                     1
O. insularis                     1
O. insularis                     1
O. insularis                     1
O. insularis                     1
O. insularis                     --
O. insularis                     --
O. insularis                     --
O. insularis                     1
O. insularis                     1
Octopus sp.          EF093793 ([dagger])
Octopus vulgaris     AJ390315 ([dagger])
O. vulgaris                      1
O. vulgaris                      1
O. vulgaris                      1
O. vulgaris                      1
O. vulgaris                      3
O. vulgaris                      --
O. vulgaris                      1
O. vulgaris                      1
O. vulgaris                      3
O. vulgaris                      1
O. vulgaris                      3
O. vulgaris                      1
O. vulgaris                      --
O. vulgaris                      --
O. vulgaris                      3
O. vulgaris                      1
O. vulgaris                      1
O. vulgaris                      1
O. vulgaris                      1
O. vulgaris                      1
O. vulgaris                      1
O. vulgaris                      --
O. vidgaris                      --
O. vidgaris                      1
O. vidgaris                      1
O. vulgaris                      1

O. vulgaris                      3

O. vulgaris                      --

O. vulgaris                      1

O. vidgaris                      1
O. vulgaris                      --
O. vulgaris                      --
O. vidgaris                      --
O. vulgaris                      3
O. vulgaris                      3
O. vulgaris          AJ390317 ([dagger])
O. vulgaris          AJ252771 ([dagger])
O. varlgaris         AJ616307 ([dagger])
O. vidgaris          AJ616308 ([dagger])
O. vidgaris          AJ390314 ([dagger])
O. vidgaris          AJ390316 ([dagger])
O. vulgaris *        AJ252770 ([dagger])
O. vulgaris          DQ683247 ([dagger])
O. vulgaris          DQ683248 ([double dagger])
O. vidgaris          DQ683249 ([double dagger])
O. vulgaris          AJ390310 ([dagger])
O. vidgaris          DQ683234 ([dagger])

O. vulgaris          DQ683235 ([dagger])

O. vulgaris          DQ683236 ([dagger])

O. vulgaris          DQ683237 ([dagger])

O. vulgaris          DQ683238 ([dagger])

O. vulgaris          DQ683239 ([dagger])

O. vulgaris          DQ683250 ([dagger])
O. vulgaris          DQ683228 ([dagger])

O. vulgaris          DQ683229 ([dagger])

O. vulgaris          DQ683232 ([dagger])

O. vulgaris          DQ683233 ([dagger])

O. vulgaris          DQ683230 ([dagger])

O. vulgaris          DQ683231 ([dagger])

O. vulgaris          DQ683240 ([dagger])

O. vulgaris          AJ390312 ([dagger])

O. vulgaris          DQ683244 ([dagger])
O. vulgaris          DQ683245 ([dagger])
O. vulgaris          DQ683246 ([dagger])
O. vulgaris          DQ683241 ([dagger])
O. vulgaris          DQ683242 ([double dagger])
O. vulgaris          DQ683243 ([dagger])
O. vulgaris          AJ616309 ([dagger])
O. vulgaris                      --

O. vulgaris                      --
O. vulgaris                      --

O. vulgaris                      --
O. vulgaris          AJ252777 ([dagger])
O. vulgaris          AJ252778 ([dagger])
O. vulgaris          AJ252773 ([dagger])
O. vulgaris                      --
Octopus              AJ390321 ([section])
  bimaculoides
Octopus              AJ390222 ([section])
  californicus
Octopus mimus        AJ390918 ([section])/
                       AJ390919 ([section])

Octopus maya                     --
Octopus hummelinck               2
Amphioctopus sp.                 2
Eledone massyae                  2

Aapalochlaena        AY545107 ([section])
  maculosa

     Species                    COI                     Origin

Octopus insularis                --               Cabo Norte-AP
O. insularis                     --               Cabo Norte-AP
O. insularis                     --               Braganca-PA
O. insularis                     1                Braganca-PA
O. insularis                     1                Braganca-PA
O. insularis                     1                Fortaleza-CE
O. insularis                     1                Fortaleza-CE
O. insularis                     --               Fortaleza-CE
O. insularis                     1                Fortaleza-CE
O. insularis                     1                Natal-RN
O. insularis                     1                Natal-RN
O. insularis                     1                Rio Grande
                                                    do Norte
O. insularis                     1                Natal-RN
O. insularis                     1                Natal-RN
O. insularis                     1                Rio Grande
                                                    do Norte
O. insularis                     1                Natal-RN
O. insularis                     1                Natal-RN
O. insularis                     1                Natal-RN
O. insularis                     1                Natal-RN
O. insularis                     1                Natal-RN
O. insularis                     1                Natal-RN
O. insularis                     1                Natal-RN
O. insularis                     1                Natal-RN
O. insularis                     --               Natal-RN
O. insularis                     1                Baia da Traico-PB
O. insularis                     1                Baia da Traico-PB
O. insularis                     1                Recife-PE
O. insularis                     1                Recife-PE
O. insularis                     1                Barra Grande-BA
O. insularis                     2                Barra Grande-BA
O. insularis                     1                Barra Grande-BA
O. insularis                     1                Salvador-BA
O. insularis                     --               Salvador-BA
Octopus sp.                      --               Natal-RN
Octopus vulgaris                 --               Recife-PE
O. vulgaris                      1                Cabo Norte-AP
O. vulgaris                      --               Braganca-PA
O. vulgaris                      1                Braganca-PA
O. vulgaris                      1                Braganga-PA
O. vulgaris                      3                Braganca-PA
O. vulgaris                      1                Braganca-PA
O. vulgaris                      1                Salvador-BA
O. vulgaris                      1                Rio de Janeiro-RJ
O. vulgaris                      3                Rio de Janeiro-RJ
O. vulgaris                      --               Rio de Janeiro-RJ
O. vulgaris                      --               Rio de Janeiro-RJ
O. vulgaris                      --               Rio de Janeiro-RJ
O. vulgaris                      3                Rio de Janeiro-RJ
O. vulgaris                      3                Jureia-SP
O. vulgaris                      --               Jureia-SP
O. vulgaris                      --               Jureia-SP
O. vulgaris                      3                Jureia-SP
O. vulgaris                      --               Guaruja-SP
O. vulgaris                      1                Guaruja-SP
O. vulgaris                      --               Guaruja-SP
O. vulgaris                      --               Guaratuba-PR
O. vulgaris                      1                Guaratuba-PR
O. vidgaris                      3                Paranagua-PR
O. vidgaris                      --               Paranagua-PR
O. vidgaris                      --               Paranagua-PR
O. vulgaris                      --               Cabo de Santa
                                                    Marta-SC
O. vulgaris                      --               Cabo de Santa
                                                    Marta-SC
O. vulgaris                      3                Cabo de Santa
                                                    Marta-SC
O. vulgaris                      1                Cabo de Santa
                                                    Marta-SC
O. vidgaris                      1                Portugal
O. vulgaris                      --               Portugal
O. vulgaris                      --               Portugal
O. vidgaris                      3                Portugal
O. vulgaris                      --               Portugal
O. vulgaris                      --               Portugal
O. vulgaris                      --               Taiwan
O. vulgaris                      --               Taiwan
O. varlgaris                     --               Japan
O. vidgaris                      --               Rio de Janeiro-RJ
O. vidgaris                      --               Itajai-SC
O. vidgaris                      --               Venezuela
O. vulgaris *                                     Venezuela
O. vulgaris             DQ683221 ([dagger])       Galicia, Spain
O. vulgaris          DQ683222 ([double dagger])   Galicia-Spain
O. vidgaris          DQ683223 ([double dagger])   Galicia, Spain
O. vulgaris                      --               France
O. vidgaris             DQ683214 ([dagger])       Durban, South
                                                    Africa
O. vulgaris          DQ683215 ([double dagger])   Durban, South
                                                    Africa
O. vulgaris             DQ683216 ([dagger])       Durban, South
                                                    Africa
O. vulgaris             DQ683217 ([dagger])       Durban, South
                                                    Africa
O. vulgaris          DQ683218 ([double dagger])   Durban, South
                                                    Africa
O. vulgaris          DQ683219 ([double dagger])   Durban, South
                                                    Africa
O. vulgaris             DQ683227 ([dagger])       Mediterranean
O. vulgaris             DQ683212 ([dagger])       Porto Elizabeth,
                                                    South Africa
O. vulgaris          DQ683213 ([double dagger])   Porto Elizabeth,
                                                    South Africa
O. vulgaris             DQ683210 ([dagger])       Struisbaai, South
                                                    Africa
O. vulgaris             DQ683211 ([dagger])       Struisbaai, South
                                                    Africa
O. vulgaris             DQ683208 ([dagger])       Hout Bay,
                                                    South Africa
O. vulgaris             DQ683209 ([dagger])       Hout Bay,
                                                    South Africa
O. vulgaris             DQ683220 ([dagger])       Umhlanga, South
                                                    Africa
O. vulgaris                      --               False Bay,
                                                    South Africa
O. vulgaris          DQ683224 ([double dagger])   Senegal
O. vulgaris          DQ683225 ([double dagger])   Senegal
O. vulgaris             DQ683226 ([dagger])       Senegal
O. vulgaris          DQ683205 ([double dagger])   Tristan da Cunha
O. vulgaris          DQ683206 ([double dagger])   Tristan da Cunha
O. vulgaris             DQ683207 ([dagger])       Tristan da Cunha
O. vulgaris                      --               Greece
O. vulgaris             FN424381 ([dagger])       Saint Paul
                                                    Islands, Southern
                                                    Indian Ocean
O. vulgaris             AB191269 ([dagger])       Japan
O. vulgaris             AB430548 ([dagger])       Japan

O. vulgaris             AB052253 ([dagger])       Japan
O. vulgaris                      --               France
O. vulgaris                      --               France
O. vulgaris                      --               Tenerife
O. vulgaris                      --               France
Octopus                 AF377967 ([section])      Santa Barbara,
  bimaculoides                                      USA
Octopus                 AF377968 ([section])      Santa Barbara,
  californicus                                      USA
Octopus mimus                    --               Iquique, Chile;
                                                    Isla de Cocos,
                                                    Costa Rica
Octopus maya            GU362545 ([section])      Mexico
Octopus hummelinck               2                Ceara
Amphioctopus sp.                 2                Para
Eledone massyae                  2                Cassino, Rio Grande
                                                    do Sul
Aapalochlaena           AB430531 ([section])      Unknown, Taiwan
  maculosa

     Species                    Reference                 Code

Octopus insularis    Current study                     OinAP 25
O. insularis         Current study                     OinAP 26
O. insularis         Current study                     OinPA3
O. insularis         Current study                     OinPA10
O. insularis         Current study                     OinPA14
O. insularis         Current study                     OinCE09
O. insularis         Current study                     OinCE16
O. insularis         Current study                     OinCE36
O. insularis         Current study                     OinCE38
O. insularis         Current study                     OinRNl
O. insularis         Current study                     OinRN2
O. insularis         Current study                     OinRN04

O. insularis         Current study                     OinRN5
O. insularis         Current study                     OinRN7
O. insularis         Current study                     OinRN08

O. insularis         Current study                     OinRN12
O. insularis         Current study                     OinRN23
O. insularis         Current study                     OinRN24
O. insularis         Current study                     OinRN25
O. insularis         Current study                     OinRN26
O. insularis         Current study                     OinRN30
O. insularis         Current study                     OinRN34
O. insularis         Current study                     OinRN56
O. insularis         Current study                     OinRN245
O. insularis         Current study                     OinPB266
O. insularis         Current study                     OinPB268
O. insularis         Current study                     OinPEl
O. insularis         Current study                     OinPE2
O. insularis         Current study                     OinBA05
O. insularis         Current study                     OinBA06
O. insularis         Current study                     OinBA125
O. insularis         Current study                     OinBA199
O. insularis         Current study                     OinBA200
Octopus sp.          Leite et al. (2008)               OinRNW
Octopus vulgaris     Warnke et al. (2004)              OinPE
O. vulgaris          Current study                     OvuAP225
O. vulgaris          Current study                     OvuPA1
O. vulgaris          Current study                     OvuPA173
O. vulgaris          Current study                     OvuPA78
O. vulgaris          Current study                     OvuPA79
O. vulgaris          Current study                     OvuPA184
O. vulgaris          Current study                     OvuBA117
O. vulgaris          Current study                     OvuRJ130
O. vulgaris          Current study                     OvuRJ131
O. vulgaris          Current study                     OvuRJ214
O. vulgaris          Current study                     OvuRJ219
O. vulgaris          Current study                     OvuRJ220
O. vulgaris          Current study                     OvuRJ280
O. vulgaris          Current study                     OvuSP24
O. vulgaris          Current study                     OvuSP40
O. vulgaris          Current study                     OvuSP41
O. vulgaris          Current study                     OvuSP306
O. vulgaris          Current study                     OvuSP307
O. vulgaris          Current study                     OvuSP308
O. vulgaris          Current study                     OvuSP310
O. vulgaris          Current study                     OvuPR2
O. vulgaris          Current study                     OvuPR3
O. vidgaris          Current study                     OvuPR43
O. vidgaris          Current study                     OvuPR45
O. vidgaris          Current study                     OvuPR46
O. vulgaris          Current study                     OvuSC5

O. vulgaris          Current study                     OvuSC6

O. vulgaris          Current study                     OVUSC10

O. vulgaris          Current study                     OVUSC11

O. vidgaris          Current study                     OvuPT5
O. vulgaris          Current study                     OvuPT11
O. vulgaris          Current study                     OvuPT12
O. vidgaris          Current study                     OvuPT13
O. vulgaris          Current study                     OvuPT37
O. vulgaris          Current study                     OvuPT40
O. vulgaris          Warnke et al. (2004)              OvuTW
O. vulgaris          Hundelot (unpubl.)                OvuTW2
O. varlgaris         Warnke et al. (2004)              OvuJP
O. vidgaris          Warnke et al. (2004)              OvuRJ
O. vidgaris          Warnke et al. (2004)              OvuSC
O. vidgaris          Warnke et al. (2004)              OvuVE
O. vulgaris *        Hudelot (unpubl.)                 OvuVE2
O. vulgaris          Teske et al. (2007)               OvuGA1
O. vulgaris          Teske et al. (2007)               OvuGA2
O. vidgaris          Teske et al. (2007)               OvuGA4
O. vulgaris          Warnke et al. (2004)              OvuFR
O. vidgaris          Teske et al. (2007)               OvuDB1

O. vulgaris          Teske et al. (2007)               OvuDB2

O. vulgaris          Teske et al. (2007)               OvuDB3

O. vulgaris          Teske et al. (2007)               OvuDB4

O. vulgaris          Teske et al. (2007)               OvuDB5

O. vulgaris          Teske et al. (2007)               OvuDB6

O. vulgaris          Teske et al. (2007)               OvuME1
O. vulgaris          Teske et al. (2007)               OvuPE2

O. vulgaris          Teske et al. (2007)               OvuPE3

O. vulgaris          Teske et al. (2007)               OvuSB5

O. vulgaris          Teske et al. (2007)               OvuSB6

O. vulgaris          Teske et al. (2007)               OvuHB3

O. vulgaris          Teske et al. (2007)               OvuHB4

O. vulgaris          Teske et al. (2007)               OvuUM1

O. vulgaris          Warnke et al. (2004)              OvuFB

O. vulgaris          Teske et al. (2007)               OvuSE1
O. vulgaris          Teske et al. (2007)               OvuSE2
O. vulgaris          Teske et al. (2007)               OvuSE4
O. vulgaris          Teske et al. (2007)               OvuTC2
O. vulgaris          Teske et al. (2007)               OvuTC3
O. vulgaris          Teske et al. (2007)               OvuTC5
O. vulgaris          Warnke et al. (2004)              OvuGC
O. vulgaris          Guerra et al. (2010)              OVUSIO

O. vulgaris          Takumiya et al. (2005)            OvuJP2
O. vulgaris          Kaneko and Kubodera               OvuJP3
                       (unpubl.)
O. vulgaris          Minataka et al. (unpubl.)         OvuJP4
O. vulgaris          Hudelot (unpubl.)                 OvuFR2
O. vulgaris          Hudelot (unpubl.)                 OvuFR3
O. vulgaris          Hudelot (unpubl.)                 OvuTE
O. vulgaris          Allcock et al. (2006)             OvuFR4
Octopus              Warnke et al. (2004),             ObimUS
  bimaculoides         Carlini et al. (2001)
Octopus              Warnke et al. (2004),             OcalUS
  californicus         Carlini et al. (2001)
Octopus mimus        Warnke et al. (2004)              OmimCR/
                                                         OmimCH

Octopus maya         Juarez et al. (unpubl.)           OmayMX
Octopus hummelinck   Current study                     OhumCE1
Amphioctopus sp.     Current study                     AmspPA86
Eledone massyae      Current study                     EmasRS53

Aapalochlaena        Strugnell et al. (2004),          Hmac/Hmac/
  maculosa             Kaneko and Kubodera               Hmac
                       (unpubl.)

* Specimens of unknown geographical origin (GenBank records).

([dagger]) Specimens included in the phylogeographical analysis only.

([double dagger]) Specimens included in the population analysis only.

([section]) Specimens included in the phylogenetic analysis only.

APPENDIX B
Specimens in which the different 16S rDNA haplotypes
identified in the current study were recorded.

Haplotype    n                   Code                      Origin

Hap_1        13   OvuAP_225, OvuPA_1, OvuPA_78,        America
                    OvuPA_79, OvuRJ_220 OvuRJ_131,
                    OvuSP_308, OvuSP_310, OvuPR_2
                    OvuPR_45 OvuPR_46 OvuSC_6, OvuSC
Hap_2         1   OvuPA_173                            America
Hap_3         1   OvuBA_117                            America
Hap_4         3   OvuRJ_130 OvuSP_41 OvuSC_5           America
Hap_5         5   OvuRJ_219 OvuRJ_214 OvuSP_40         America
                    OvuSP_306 OvuSP_41
Hap_6         1   OvuSC_11                             America
Hap_7         1   OvuVE                                America
Hap_8         1   OvuVE_2                              America
Hap_9         7   OvuPT_37, OvuPT_40, OvuPT_5,         Europe, Africa
                     OvuGA_4, OvuGA_2OvuGA_1 OvuFR
Hap_10        1   OvuPT_13                             Europe, Africa
Hap-11        1   OvuME                                Europe, Africa
Hap_12        1   OvuSE_4                              Europe, Africa
Hap_13        1   OvuSE_2                              Europe, Africa
Hap_14        1   OvuSE_1                              Europe, Africa
Hap-15        2   OvuTC_5, OvuTC_3                     Europe, Africa
Hap_16        1   OvuTC_2                              Europe, Africa
Hap_17        2   OvuUM_1, OvuDB_2                     Europe, Africa
Hap_18        1   OvuDB_6                              Europe, Africa
Hap_19        3   OvuDB_5 OvuSB_5 OvuFB                Europe, Africa
Hap_20        3   OvuDB_4 OvuHB_3 OvuPE_2              Europe, Africa
Hap_21        1   OvuDB_3                              Europe, Africa
Hap_22        1   OvuDB_1                              Europe, Africa
Hap_23        2   OvuSB_6, OvuPE_3                     Europe, Africa
Hap_24        1   OvuHB_4                              Europe, Africa
Hap_25        1   OvuGC                                Europe, Africa
Hap_26        1   OvuFR_2                              Europe, Africa
Hap_27        1   OvuFR_3                              Europe, Africa
Hap_28        1   OvuTE                                Europe, Africa
Hap_29        1   OvuTW                                Asia
Hap_30        1   OvuTW_2                              Asia
Hap_31        1   OvujP                                Asia

APPENDIX C
Specimens in which the different COI haplotypes identified in the
current study were recorded.

Haplotype    n                 Code                       Origin

Hap_1        12   OvuAP_225, OvuPA_78, OvuPA_79,   America
                    OvuRJ_130, OvuRJ_131,
                    OvuRJ_280, OvuSP_308,
                    OvuSP_306, OvuSP_24,
                    OvuPR_3, OvuPR_43, OvuSC_11
Hap_2         1   OvuPA_184                        America
Hap_3         1   OvuPA_173                        America
Hap_4         1   OvuBA_117                        America
Hap_5         1   OvuSC_10                         America
Hap_6         6   OvuPT_13, OvuPT_5, OvuPT_12,     Europe, Africa
                    OvuGA_4, OvuGA_2, OvuGA_1
Hap_7        17   OvuME_1, 0vuSE_4, OvuSE_2,       Europe, Africa, Asia
                    OvuSE_1, OvuUM_1, OvuDB_6,
                    0vuDB_5, 0vuDB_4, 0vuDB_2,
                    0vuPE_3, 0vuPE_2, OvuSB_5,
                    0vuHB_3, OvuTC_5, OvuTC_3,
                    OvuTC_2, OvuSIO
Hap_8         1   OvuDB_3                          Europe, Africa
Hap_9         1   OvuDB_1                          Europe, Africa
Hap_10        1   OvuSB_6                          Europe, Africa
Hap_11        1   OvuHB_4                          Europe, Africa
Hap_12        1   OvuJP_2                          Asia
Hap_13        1   0vuJP_3                          Asia
Hap_14        1   OvuJP_4                          Asia


ACKNOWLEDGMENTS

We especially thank the owners of commercial fisheries in Braganca (Para) for the donation of the specimens analyzed in this study, and Edimario Fernandes da Cruz for providing specimens from northeastern Brazil. We are also grateful to Dr. Marcelo Vallinoto for his help with the Haploview program. In particular, we thank Celia Ferreira Alencar (in memoriam) for her valuable personal friendship and professional assistance with the sequencing reactions conducted by J.B.S. during the initial stages of this project, without which the current study would not have been impossible. This study was funded by CNPq through CT-Hidro project no. 552126/2005-5 (masters scholarship to J.B.S.) and research grants to I.S. (308477/2006-5) and H.S. (300741/2006-5).

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JOAO BRAULLIO DE LUNA SALES, (1) * PERICLES SENA DO REGO, (1) ALEXANDRE WAGNER S. HILDORF, (2) ANGELA A. MOREIRA, (2,3) MANUEL HAIMOVICI, (4) ACACIO R. TOMAS, (5) BRUNO B. BATISTA, (6) REYNALDO A. MARINHO, (6) UNAI MARKAIDA, (7) HORACIO SCHNEIDER, (1) IRACILDA SAMPAIO (1)

(1) Universidade Federal do Para, Campus Universitario de Braganca, Laboratorio de Genetica e Biologia Molecular, Braganca/PA CEP 68600-000, Brazil; (2) Universidade de Mogi das Cruzes, Nucleo Integrado de Biotecnologia, Laboratorio de Genetica de Peixes e Aquicultura, Mogi das Cruzes/SP CEP 08780-911, Brazil; (3) Universidade de Sao Paulo, Programa de Pos-Graduacao Interunidades em Biotecnologia, Sao Paulo/SP, CEP 05508-000, Brazil: (4) Universidade Federal do Rio Grande, Laboratorio de Recursos Demersais e Cefalopodes, Caixa Postal 474, Rio Grande/RS CEP 96201-900, Brazil; (5) Instituto de Pesca Centro de Pesquisa Pesqueira Marinha, Santos/SP, CEP 11030-906, Brasil; (6) Universidade Federal do Ceara, Laboratorio de Biologia e Tecnologia Pesqueira, Fortaleza/CE, CEP 60455-460, Brazil; (7) Linea de Pesquerias Artesanales, El Colegio de la Frontera Sur, CONAC YT, Calle 264, 24000 Campeche, Mexico

* Corresponding author. E-mail: braziliancephalopod@gmail.com

DOI: 10.2983/035.032.0211

TABLE 1.
Primers used for the PCR amplification of the 2 genes
analyzed in the current study.

Gene             Primers               References

16S    5'-GCCTGCCTGTTTACCAAAAAC-3'   Palumbi et al.
       5'-CGGTCTGAACTCAGATCACGT-3'     (1991)
COI    5'-GGTCAAACAAATCATAAAGA       Folmer et al.
         TATTGG-3'                     (1994)
       5'-TAAAATTCAGGGTGACCAAAA
         AATCA-3'

TABLE 2.
Genetic divergence between among species groups identified
through the analysis of the 16S rDNA gene.

                                      Group

      Group           1        2        3        4        5

Group 1                        0.8^     0.8^     3.3^     4.0^
Group 2               2.0*              0.7^     3.1^     3.7^
Group 3               2.0*     1.6*              2.7^     3.6^
Octopus               9.7*     9.1*     7.8*              3.8^
  bimcauloides
Octopus insularis    11.2*     9.7*    10.1*    11.2*
Octopus mimus        12.9*    12.3*    11.7*    10.1*     5.3*
Octopus              13.2*    13.5*    13.5*    10.4*    13.2*
  hummelincki
Amphioctopus sp.     14.9*    15.3*    14.8*    15.8*    13.6*
Hapalochlaena        19.0*    18.6*    18.5*    20.9*    17.2*
  maculosa
Eledone massyae      26.4*    25.9*    26.4*    22.5*    24.5*

                                      Group

      Group           6        7        8        9       10

Group 1               5.1^     5.7^    11.2^    16.1^    17.6^
Group 2               4.9^     5.6^    11.3^    16.1^    17.2^
Group 3               4.6^     6.0^    11.1^    16.6^    17.7^
Octopus               3.4^     3.6^     9.5^    18.1^    17.3^
  bimcauloides
Octopus insularis     1.8      4.5^     5.8^     8.7^    17.8^
Octopus mimus                  6.2^     9.1^    12.3^    18.7^
Octopus              16.5*              6.1^    12.8^    17.2^
  hummelincki
Amphioctopus sp.     16.1*    13.8*              5.1^    15.1^
Hapalochlaena        19.6*    19.4*    10.4*             11.1^
  maculosa
Eledone massyae      26.0*    24.1*    19.1*    18.4*

The values in bold type (below the diagonal) are the nucleotide
divergence values (percent); values in italics (above the
diagonal) are SDs.

Note: Values in bold type (below the diagonal) are the nucleotide
divergence values (percent) indicated with *.

Note: Values in italics (above the diagonal) are SDs indicated with ^.

TABLE 3.
Genetic divergence among the species groups identified
through the analysis of the COI gene.

                                      Group

      Group           1        2        3        4        5

Group 1                        0.7^     0.7^     2.6^     2.2^
Group 2               2.6*              0.6^     2.8^     2.3^
Group 3               3.2*     2.6*              2.7^     2.3^
Octopus              16.9*    18.8*    18.3*              1.9^
  bimcauloides
Octopus insularis    13.7*    14.8*    15.0*    11.2*
Octopus mimus        17.8*    18.8*    18.9*    12.8*     8.6*
Octopus              15.1*    15.2*    16.6*    17.8*    16.3*
  hummelincki
Amphioctopus sp.     20.2*    20.1*    20.3*    23.3*    22.0*
Hapalochlaena        21.1*    21.7*    22.8*    23.3*    23.0*
  maculosa
Eledone massyae      19.9*    19.2*    20.6*    22.0*    22.0*

                                      Group

      Group           6        7        8        9       10

Group 1               2.6^     2.3^     3.2^     3.2^     2.8^
Group 2               2.7^     2.4^     3.2^     3.2^     2.8^
Group 3               2.7^     2.5^     3.1^     3.3^     2.9^
Octopus               2.0^     2.7^     3.4^     3.4^     3.3^
  bimcauloides
Octopus insularis     1.4^     2.5^     3.3^     3.4^     3.2^
Octopus mimus                  2.6^     3.6^     3.7^     3.2^
Octopus              18.3*              3.2^     3.0^     2.8^
  hummelincki
Amphioctopus sp.     24.4*    21.5*              2.8^     3.2^
Hapalochlaena        25.1*    19.6*    18.7*              3.0^
  maculosa
Eledone massyae      22.6*    19.3*    22.2*    20.8*

The values in bold type (below the diagonal) are the nucleotide
divergence  values (percent); values in italics (above the
diagonal) are SDs.

Note: The values in bold type (below the diagonal) are the
nucleotide divergence  values (percent) indicated with *.

Note: Values in italics (above the diagonal) are SDs indicated with ^.

TABLE 4.
Diversity indices derived from the sequences of the 16S rDNA
gene analyzed for the different Octopus vulgaris populations
analyzed in the current study.

Group     n    PS        H              Pi         Tajima's D   Fu's Fs

Group 1   27   10   0.85 (0.042)   0.005 (0.000)     -0.468     -1.686
Group 2   33   10   0.81 (0.047)   0.005 (0.006)     -1.359     -2.021
Group 3    3    5   1.00 (0.272)   0.007 (0.002)     0.000       0.587
Total     63   24   0.90 (0.021)   0.014 (0.000)     -0.451     -2.887

N, number of individuals; PS, polymorphic sites; [H.sub.2] haplotype
diversity; Pi, nucleotide diversity; Tajima's D, value of Tajima's
statistics; Fu's Fs, Value of Fu's statics; PS, polymorphic sites.
Standard deviation values are in parenthesis.

TABLE 5.
Results of the analysis of molecular variance and the fixation
index ([F.sub.st] for the 16S rDNA gene in populations of Octopus
vulgaris.

                                        16S
Octopus vulgaris
Source of the variation   % of Variation   [F.sub.st]

Between populations           62.44          0.62 *
Within populations            37.56

* Significant P < 0.05.

TABLE 6.
Estimates of genetic differentiation among Octopus vulgaris
populations based on the [PHI]st values for the mitochondrial 16S
rDNA gene.

          Group 1   Group 2

Group 2   0.640 *     --
Group 3   0.811 *   0.511

* P < 0.05.

TABLE 7.
Diversity indices derived from the sequences of the COI gene
analyzed for the different Octopus vulgaris populations analyzed
in the current study.

  Group     n    PS         H              Pi

Group 1     16   14   0.45 (0.151)    0.004 (0.002)
Group 2     26    9   0.58 (0.093)    0.004 (0.001)
Group 3      4   20   1.00 (0.177)    0.021 (0.006)
Ovu total   46   35   0.79 (0.042)    0.016 (0.001)

  Group       Tajima's D        Fu's Fs

Group 1     -1.976 (0.921)   0.408 (1.640)
Group 2     -1.999 (0.004)   0.158 (1.047)
Group 3     -0.697 (0.921)   0.353 (0.626)
Ovu total   -0.133           1.296

N, number of individuals; PS, polymorphic sites; H, haplotype
diversity; Pi, nucleotide diversity; Tajima's D, value of
Tajima's statistics; Fu's Fs,  value of Fu's statistics; PS,
polymorphic sites. Standard deviation values are in parenthesis.

TABLE 8.
Results of the analysis of molecular variance and the fixation
index ([F.sub.st]) for the COI gene in populations of Octopus
vulgaris.

O. vulgaris                             COI
Source of the
variation             % of the Variation   [F.sub.st]

Between populations         79.00            0.79 *
Within populations          21.00

* Significant P < 0.05.

TABLE 9.
Estimates of genetic differentiation among Octopus vulgaris
populations based on the [PHI]st values for the mitochondrial COI
gene.

          Group 1   Group 2

Group 2   0.838 *     --
Group 3   0.739 *   0.697 *

* P < 0.05.
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Author:Sales, Joao Braullio De Luna; Do Rego, Pericles Sena; Hildorf, Alexandre Wagner S.; Moreira, Angela
Publication:Journal of Shellfish Research
Article Type:Report
Geographic Code:1MEX
Date:Aug 1, 2013
Words:8692
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