Phylogeographical features of octopus vulgaris and octopus insularis in the Southeastern Atlantic based on the analysis of mitochondrial markers.
KEY WORDS: phylogeography, Octopus vulgaris, Octopus insularis, South Atlantic, genetics, mitochondrial DNA
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 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).
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).
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.
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
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: email@example.com
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|
|Date:||Aug 1, 2013|
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