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Close genetic relationships between two American octopuses: Octopus hubbsorum berry, 1953, and Octopus mimus Gould, 1852.

ABSTRACT The octopuses Octopus hubbsorum and Octopus mimus are two species of octopuses that inhabit shallow waters of the eastern Pacific Ocean. The former species is found in the north, from the Gulf of California to Oaxaca, Mexico, whereas O. mimus lives in the south, from Peru to Chile. To infer the phylogenetic relationships between these species we used the mitochondrial genes cytochrome oxidase subunits I and III, and r16S with the aim of comparing their genetic distances with those of other Octopus sp. The genetic distance values between both species estimated per gene or concatenated were low (0%- 1.6%) in comparison with other interspecific distances values (4.6%-18.4%). Application of 10x rule showed there is no overlap between intra- and interspecific octopus distances, whereas the application of the 4x rule confirmed that both octopuses belong to the same lineage. The resolved topologies with maximum parsimony and Bayesian approaches clustered the specimens of O. hubbsorum and O. mimus in a single clade with high bootstrap and posterior probability values (100 and 1.0, respectively). These results allow us to suggest that O. hubbsorum and O. mimus could represent the same species.

KEY WORDS: Octopodidae, taxonomy, mitochondrial DNA, synonymy, Octopus hubbsorum, Octopus mimus

INTRODUCTION

The taxonomy of the Octopodidae is complex. Of the 374 nominal species listed in Norman and Hochberg (2005), only 186 are considered as valid and 55 are uncertain. The lack of hard structures and the soft bodies of octopods, which often are poorly preserved and, as a result, badly distorted after death, plus the high phenotypic plasticity they exhibit, which allows them to change their body pattern in color and texture as an expression of their behavior, lead teuthologists to use molecular data to discriminate taxa, find cryptic species in several regions of the world, and place uncertain specimens in the correct genus or species (Soller et al. 2000, Warnke et al. 2002, Allcock et al. 2007, Leite et al. 2008, Undheim et al. 2010, Kaneko et al. 2011, Toussaint et al. 2012).

The octopuses Octopus hubbsorum Berry, 1953, and Octopus mimus Gould, 1852, are two species of octopuses that inhabit the shallow waters of the eastern Pacific Ocean. The former is found in the north, where it is reported to range from the Gulf of California to Oaxaca, Mexico (Lopez-Uriarte et al. 2005); O. mimus lives in the south and ranges in distribution from north of Peru to Chile (Guerra et al. 1999). Both species are the main targets of octopus fisheries in their respective geographic areas. However, the distribution boundaries of these two species are not well documented. Lopez-Uriarte et al. (2005) pointed out the presence of O. hubbsorum farther south of the Mexican coast whereas Guerra et al. (1999) argued that more specimens need to be analyzed to define the distribution of O. mimus. Recently, the geographic distribution range of O. hubbsorum has been extended (Dominguez-Contreras et al. 2013) and, at the beginning of the past decade, molecular studies indicated that O. mimus was present in Central America (Soller et al. 2000, Warnke et al. 2002). Current data suggest the presence of O. mimus off the Pacific coast of Mexico (Flores-Valle et al. unpubl. data).

The phylogenetic relationships of Octopus hubbsorum are unknown whereas those of Octopus mimus have drawn little attention. For decades it had been considered that the principal species inhabiting the South American Pacific was Octopus vulgaris, and it was not until 1995 and 1999, that Cortez and Warnke, respectively, indicated that O. mimus was the species captured along the Chilean and Peruvian coasts. Subsequently, molecular studies revealed the distinction between O. mimus and O. vulgaris (Soller et al. 2000, Perez-Losada et al. 2002, Warnke et al. 2002). These works also described a close relationship between O. mimus and two Atlantic species, Octopus insularis Leite and Haimovici, 2008, and Octopus may a Voss and Solis-Ramirez, 1966. Nevertheless, the relationship of O. mimus with other species from the American Pacific is not known; thus, the aim of the current study is to define the phylogenetic relationship between O. hubbsorum and O. mimus based on 3 mitochondrial molecular markers, cytochrome oxidase subunit I (COI), cytochrome oxidase subunit III (COIII), and r16S. This study will help to clarify the taxonomy of these two morphologically similar species.

MATERIALS AND METHODS

Sampling

Specimens of one octopus species identified as Octopus hubbsorum were captured in several localities off the Pacific coast of Mexico (Fig. 1). Identifications were based on the key in Roper et al. (1995) for cephalopods from the American Central Pacific and the original description of O. hubbsorum in Berry (1953), and by comparison of voucher specimens of O. hubbsorum deposited in the malacological collection of the Santa Barbara Museum of Natural History.

An arm tissue sample was taken from each octopus and stored in 70% ethyl alcohol until DNA extraction. The remainder of each octopus specimen was fixed in 10% formaldehyde and then preserved in 70% ethyl alcohol to be deposited as voucher specimens in the malacological collection of the Escuela Nacional de Ciencias Biologicas of the Instituto Politecnico Nacional. In addition, tissue samples of two unidentified Octopus specimens from Colombia and one of Octopus bimaculoides Pickford and McConnaughey, 1949, were donated by the Santa Barbara Museum of Natural History. The sequences of Octopus mimus included in this study were 4 for COI, 6 for COIII, and 2 for r16S, and these were taken from Soller et al. (2000), Warnke et al. (2002, 2004), and Acosta-Jofre et al. (2012). In addition, partial sequences for these 3 genes were obtained from GenBank for 7 taxa, including the outgroup taxon Opisthoteuthis sp. (see Appendix A for accession numbers).

DNA Extraction, Amplification, and Sequencing

Total DNA was extracted and purified with the kit Wizard SV Genomic DNA Purification System (Promega) according to the manufacturer's protocol. The primers used were those indicated in the references listed in Table 1. Polymerase chain reaction (PCR) was used for the amplification of genomic DNA. Each 25-[micro]L reaction contained 2 mM Mg[Cl.sub.2] (Qiagen), 1x PCR buffer (Qiagen), 0.4 mM dNTPs, 0.75 U Taq DNA polymerase (Promega), and 0.4 [micro]M of each primer. The thermal cycles included 1 initial cycle at 94[degrees]C for 5 min, followed by 30 cycles at 94[degrees]C for 45 sec, 38-52[degrees]C (according to the primers used; Table 1) for 45 sec, and 72[degrees]C for 90 sec. The amplified products were purified with Qiaquick PCR Purification Kit (Qiagen) according to the manufacturer's protocol.

The purified PCR product was sequenced in both directions with the BigDye Terminator v3.1 Cycle Sequencing Kit on an ABI PRISM-3100 Avant automated sequencer at the Universidad Autonoma Metropolitana (Distrito Federal, Mexico). DNA sequences were aligned with Clustal W (Thompson et al. 1994) implemented in MEGA 5.0 (Tamura et al. 2011). The alignments of the sequences were verified with the respective translation of amino acids for COI and COIII. No gaps were included in the alignment of COI and COIII.

Data Analysis and Genetic Distances

Nucleotide saturation was tested using Xia's test (Xia et al. 2003, Xia & Lemey 2009) executed in DAMBE version 5.2.73. (Xia & Xie 2001). The sequence composition for each genetic region and the nucleotide substitutions per site at each codon position between species pairs for COI and COIII were analyzed. The genetic distances were calculated by using the Tamura-Nei model (Tamura & Nei 1993). Bootstrap support was estimated using 500 iterations. All analysis were implemented in MEGA 5.0 (Tamura et al. 2011). A Mann-Whitney U test was carried out in Statistica 10 to detect significant genetic differences between inter- and intraspecific Tamura-Nei distances for the Octopus species analyzed. Moreover, the 10X rule (Hebert et al. 2004) was applied for COI distances because it frequently is used as DNA bar coding; the 4X rule (Birky et al. 2010) was applied for COIII distances because there are more Octopus mimus sequences available for this marker in GenBank. Both rules were implemented for discriminate species.

The Tajima test (Tajima 1989) was executed in DnaSP v5 (Librado & Rozas 2009) to elucidate whether the sequences are under selective pressure.

The chi square test for composition homogeneity of nucleotide, and the homogeneity test (Farris et al. 1995) for evaluation of the congruence between the data were implemented in PAUP* (Swofford 2002). JModeltest (Posada 2008) was used to select the best-fit model for each data set under the Akaike information criterion.

Phylogenetic Analysis

Phylogenetic reconstruction was done using the genes separately and as a concatenated data set. In the latter case, only COI and COIII were used because of the availability of sequences of Octopus mimus in GenBank for those 2 gene regions. Because the matrices of the data were less than 20 taxa, and because redundant sequences were discarded, an implicit enumeration search in the T.N.T. software (version 1.1) (Goloboff et al. 2008) was performed. The bootstrap values for clades support were generated by resampling 1,000 replicates.

Bayesian inference was performed in MrBayes3.2 (Ronquist & Huelsenbeck 2003). The analysis consisted of 2 independent runs with 4 chains for 1,000,000 generations, and sampling every 100. A total of 25% of the samples were discarded. In the case of the concatenated data 2,000,000 generations were required to minimize the SD.

RESULTS

Sequences Analyses and Codon Base Composition

Partial sequences of 24 Octopus hubbsorum, 2 Octopus sp. from Colombia, and 1 Octopus bimaculoides were obtained for each gene with lengths of 688 nucleotides for COI, 634 for COIII, and 526 for r16S, and a total of 1,848 bp. Because redundant sequences were discarded for the analyses and because homologous GenBank sequences for the Octopus species used in this study are not available for all 3 molecular markers, some sequences are available just for one; the number of sequences of the data set varied between each gene. Thus, 14 sequences were analyzed for COI, r16S, and COI + COIII, and 19 sequences for COIII (Appendix A).

Nucleotide saturation was not detected in any of the three molecular markers (Iss < Iss.c, P < 0.05). No significant values of Tajima's D were observed, indicating selective neutrality of the sequence data. All three mitochondrial gene regions analyzed in the Octopus species showed bias against G + C content, which prompted for the analysis of codon usage in COI and COIII regions.

All variable sites for the two coding genes were transitions except at position 429 for COIII. Nonsynonymous substitutions were detected between Octopus hubbsorum and Octopus mimus. A transition at position 475 of the COI sequences resulted in a substitution at the first position of codon 159, with O. hubbsorum having ATC (I) and O. mimus having GTC (V). Other nonsynonymous substitutions detected in COIII sequences were two transitions at positions 427 and 502, and a transversion at position 429. Mutations at positions 427 and 429 resulted in changes at the first and third positions of codon 143, resulting in ATC (I) for O. hubbsorum and GTA (V) for O. mimus. A mutation at position 502 resulted in a change in the first position of codon 168, with O. hubbsorum exhibiting the GAT (D) codon and O. mimus, the AAT (N) codon. In general, a bias against G at the third codon position was detected. Base composition per codon position for COI and COIII sequences are listed in Table 2.

Nucleotide site differences between Octopus hubbsorum and Octopus mimus for each gene are summarized in Tables 3 and 4. Four haplotypes for COI and COIII, and 3 for r16S were resolved from 24 specimens of O. hubbsorum whereas two haplotypes for COI and r16S, and 4 for COIII were resolved for O. mimus (some sequences of this species showed ambiguous sites, so they were excluded). The haplotypes were resolved respectively from 4, 2, and 6 sequences of O. mimus. Nucleotide variation between haplotypes of O. hubbsorum and O. mimus was low, with only 1 or 2 substitutions per sequence.

Genetic Distances

The calculated genetic distances were very similar between Octopus hubbsorum and Octopus mimus (Table 5) using the 3 genes. Low values were registered between O. hubbsorum, O. mimus specimens from Costa Rica, and the Octopus sp. from Colombia (0%-0.2%). Greater values were observed between O. hubbsorum and the O. mimus specimens from Chile and Peru (0.8% 1.4%), but these values are much lower than the interspecific distances calculated for Octopus sp. The lowest divergences between taxa for COI, COIII, and r16S were 8.9%, 6.4%, and 4.5%, respectively (Fig. 2). The average intraspecific divergence for O. hubbsorum was 0.2% for COI, 0.4% for COIII, and 0.3% for r16S. The differences between interspecific and intraspecific Tamura-Nei genetic distances of Octopus sp. with the 3 molecular markers were significant (U = 0, P < 0.05). The 10x threshold for Octopus (average intraspecific distance, 0.2%) is not enough to separate O. hubbsorum from O. mimus. The greatest COI distance value between O. hubbsorum and O. mimus specimens was 0.9%, which is less than the 2.0% threshold. The 4x rule did not support (D = 0.013) the reciprocal monophyly of O. hubbsorum ([pi] = 0.00394) and O. mimus ([pi] = 0.002, D < 4[theta]).

Phylogenetic Inferences

The evolution models that fit the data best were GTR + (I + G) (COI + COIII), TIM2 + G (COI and COIII), and TIM3 + G (r16S). Because the last two models cannot be implemented in MrBayes, we used the GTR + G model to infer the relationships. The topologies generated analyzing the concatenated genes (Fig. 3A, B) as well as the Bayesian and parsimony topologies of COI and r16S (Appendices B and C) concurred with the clustering of Octopus hubbsorum, Octopus mimus, and Octopus sp. from Colombia in a monophyletic clade, with the specimens from South America (Peru and Chile) forming 1 group and the octopuses from North and Central America (Mexico, Costa Rica, and Colombia) forming another. In the two combined data trees and the r16S Bayesian tree, Octopus maya is the sister group of the O. hubbsorum O. mimus clade. In the Bayesian COIII topology, the clade conformed by O. maya and Octopus insularis is the sister group of O. hubbsorum-O. mimus. The position of Octopus bimaculoides could not be resolved using COI and COIII separately. In the r16S topologies, O. bimaculoides is related to the clade conforming O. hubbsorum-O. mimus, O. maya, and O. insularis.

DISCUSSION

The A-T bias in the nucleotide sequences of codon regions has been reported previously for other species of octopuses, suggesting that the mitochondrial DNA is under directional selection toward A-T (Barriga-Sosa et al. 1995, Guzik et al. 2005). Also, a high transition-to-transversion ratio has been reported in closely related Octopus sp. (Barriga-Sosa et al. 1995). In species with high divergence, the transversion rate increases and erases the record of transitions (Brown et al. 1982). Only a transversion in the COIII sequence between Octopus hubbsorum and Octopus mimus was observed, suggesting they are still in a divergent process or they diverged recently.

The distances between Octopus hubbsorum and Octopus mimus were similar to intraspecific divergences calculated for other Octopus sp. The COI distance values of specimens of Octopus vulgaris from the Mediterranean ranged from 0.2%-3.9% (Keskin & Huseyin 2011); in South Africa, the intraspecific divergence, using COIII, for Octopus vulgaris ranged from 0.6%-1.5% (Oosthuizen et al. 2004). Even other genera in the family Octopodidae show similar intraspecific distances values. For COI, specimens of Enteroctopus have distance values ranging from 0.0%-0.8% (Toussaint et al. 2012); specimens of Pareledone, from 0.0%-2.5% (Undheim et al. 2010). Kaneko et al. (2011) reported a mean value of COI intraspecific variation of 0.2%, which is much lower than the mean value of interspecific divergence (2.8%) in benthic octopuses.

The genetic distances resolved between Octopus mimus and Octopus hubbsorum were significantly less than those reported for other closely related Octopus sp. calculated with the same fragments of genes used in the current study. The greatest distance value for the genes COIII and r16S between O. hubbsorum and O. mimus was much lower than the lowest distance value between other species of Octopus. The ocellate octopus species from the northeastern Pacific Ocean--Octopus bimaculatus Verril, 1883, and Octopus bimaculoides-showed a divergence of 5.5% calculated with COIII (Barriga-Sosa et al. 1995). These two species are morphologically very similar. They are distinguished primarily by egg size and hatchling habits. The hatchlings of the small-egg species, O. bimaculatus, are planktonic paralarvae whereas the hatchlings of the large-egg species, O. bimaculoides, are benthonic juveniles. Similarly, high genetic distances using r 16S have been reported for Octopus vulgaris Cuvier, 1797, and Octopus insularis (7.2%); and O. insularis and O. mimus (4.6%) (Leite et al. 2008). In the same manner, O. vulgaris and O. mimus showed high divergence with COIII (15%) (Soller et al. 2000) and with r 16S (6.7%) (Warnke et al. 2002). In the eastern Pacific, these two species have been treated as synonymous for a long time.

It has been demonstrated that COI is good marker to separate benthic octopuses (Kaneko et al. 2011). However, the use of COI as DNA bar coding has been widely discussed, and rules to discriminate species based on DNA markers have been proposed (Hebert et al. 2004, Meyer & Paulay 2005, Birky et al. 2010).

In this study, the 10x and 4x rules showed that Octopus hubbsorum and Octopus mimus are not distinct and separate species. The 10x rule (Hebert et al. 2004) showed that the interspecific distance between O. hubbsorum and O. mimus was less than 10x threshold. The accuracy of thresholds for delimiting species depends on the nonoverlapping between inter and intraspecific distances (Meyer & Paulay 2005). In the current study, there was no overlap, which suggests that both these octopuses are the same. Similarly, Dai et al. (2012), applying the 10X rule, indicated that COI is effective to discriminate among cephalopod species.

Although the 4x rule was applied initially to parthenogenetic organisms (Birky et al. 2010), it can be used for mitochondrial sequences of sexual organisms (Marrone et al. 2010, Baird et al. 2011, Kieneke et al. 2012, Marrone et al. 2013,) because it is based on the evolutionary genetic model of speciation (Birky et al. 2010). The 4x rule detected that the average distance between Octopus hubbsorum and Octopus mimus clades was less than the intraclade diversity and therefore they could not be considered separate species, but the same species, with differences that might be the result of random drift or population structure.

The topologies resolved here for Octopus hubbsorum and Octopus mimus with the 3 genes analyzed either independently or combined showed a close relationship between these 2 American Octopus sp. A monophyletic clade included O. hubbsorum, O. mimus, and the O. sp. from Colombia. Nevertheless, the resolved topologies using COIII and r16S gene regions showed a polytomy of O. mimus from Costa Rica along with O. hubbsorum specimens.

In the concatenated and Bayesian r16S topologies, Octopus maya, an endemic species in the Gulf of Mexico, was the sister group of the Octopus hubbsorum-Octopus mimus clade. Several studies have suggested that O. mimus is related closely to the Atlantic Octopus sp. For instance, Perez-Losada et al. (2002), using allozyme data, reported a close relationship between O. mimus and O. maya, and proposed that both octopuses originated from a common ancestor. This issue was discussed much earlier by Voight (198S) for morphologically similar species of octopus from both the Atlantic and Pacific oceans. Similarly, Soller et al. (2000) and Acosta-Jofre et al. (2012) considered that O. mimus and Octopus "vulgaris" from the Caribbean are related, and suggested that this could be the results of gene flow before the closure of the Panama Isthmus, and that the Caribbean and northeastern Atlantic O. " vulgaris" might be a cryptic species that represents a distinct species from the true O. vulgaris found in the Mediterranean and the northwestern Atlantic.

The results shown here are similar to those reported by Warnke et al. (2002, 2004) and Leite et al. (2008), in which they postulated that Octopus mimus is closely related to Octopus insularis from the north of Brazil. However, none of these works included Octopus maya.

Following the recommendation of Strugnell and Nishiguchi (2007), this study shows several topologies using different markers and phylogenetic analysis because, currently, it cannot be ruled out which gene and method is the best to infer phylogenetic relationships. However, we consider the concatenated Bayesian tree to be the most appropriate phytogeny to use for two reasons: (1) it considers a model of evolution that parsimony does not; and (2) the concatenated data set contains more characters (information) than genes analyzed individually.

Taxonomic Implications

The octopus Octopus mimus has been described previously to have a faint ocelli, which can be observed when the animal is alive (Guerra et al. 1999; F. Cardoso, Universidad Nacional Mayor de San Marcos, pers. comm., October 2012). The presence of ocelli in O. mimus is a controversial issue. They were not observed in the study by Warnke et al. (2002). However, F. Cardoso (Universidad Nacional Mayor de San Marcos, pers. comm., October 2012) has mentioned that octopuses, both with and without ocelli, are present in Peru. Ocelli are absent in Octopus hubbsorum, but this characteristic and diagnostic body pattern feature is present in O. mimus. In this regard, several hypotheses can be proposed. First, the sequences reported in GenBank as O. mimus and used in this study do not belong to the true O. mimus species, and they could belong to the other species without ocelli referred to by Cardoso. If this is the case, then O. hubbsorum could be found in South America. Second, the presence of ocelli is a morphological variable characteristic within species. Morphological plasticity has been documented in quite a few octopus populations (Doubleday et al. 2009, Storero et al. 2010), and 1 species could show several different color and texture body patterns (Borrelli et al. 2006, Barbato et al. 2007, Leite & Mather 2008). Third, the octopuses O. hubbsorum and O. mimus are two genetically similar species but with slight morphologically differences. Although this is rare in nature, currently it cannot be ruled out. The American and European lobsters--Homarus americanus Milne Edwards, 1837, and Homarus gamarus (Linnaeus, 1758)--respectively, are a case of 2 species that, although they are genetically similar, exhibit morphological differences (Hedgecock et al. 1977). Fourth, and last, the ocellate Octopus sp. that has been confused frequently with O. mimus is a distinct and different species.

Some studies have questioned the taxonomic identity of GenBank sequences, addressing that such sequences might correspond to different species of octopus in relation to those reported in articles in which the sequences were published (Leite et al. 2008, Undheim et al. 2010, Acosta-Jofre et al. 2012). Nevertheless, because the Octopus mimus sequences used in the current study came from different sources (Soller et al. 2000, Warnke et al. 2002, Warnke et al. 2004, Acosta-Jofre et al. 2012), and because O. mimus is the octopus collected most frequently off the coast of South America (Guerra et al. 1999, Cardoso et al. 2004), it would be expected that the sequences from GenBank are in fact from O. mimus. Thus, the possibility that Octopus hubbsorum and O. mimus are synonymous represents the continuum of a single octopus species present off both Central and South America that sustains the artisanal coastal octopus fisheries in these regions. However, comparative morphological analyses between the two octopuses would help to confirm our results. Plus, it is critical, in the future, to archive specimens in collections that voucher octopuses from which tissue samples were taken and used for DNA analyses.

APPENDIX A

List of octopus species used in this study with its
corresponding GenBank accession number and the malacological
collections record where the voucher specimens are stored.

Collection           Taxa         Specimens      Locality
record                               (n)

ENCB pending    Octopus               1       Oaxaca,
                  hubbsorum                     Mexico
ENCB pending    0. hubbsorum          1       Michoacan,
                                                Mexico
ENCB pending    0. hubbsorum          1       Nayarit 1,
                                                Mexico
ENCB pending    0. hubbsorum          2       Nayarit 2,
                                                Mexico
ENCB pending    0. hubbsorum          1       Sonora,
                                                Mexico
--              Octopus mimus         1       Costa Rica
--              O. mimus              1       Iquique, Chile
--              0. mimus              2       Coloso, Chile

--              0. mimus              2       Callao, Peru

SBMNH           Octopus sp.           1       Malpelo
  pending                                       Island,
                                                Colombia
SBMNH           Octopus               1       California
  pending         bimaculoides
--              Octopus               1       USA
                  californicus
--              Octopus               2       Brazil
                  insularis
--              Octopus maya          2       Yucatan,
                                                Mexico
--              Octopus               1       Brazil
                  vulgaris
--              O. "vulgaris"         1       Venezuela
--              0. "vulgaris"         1       India
--              Opisthoteuthis        1       South Africa
                  sp.
--              Scaeurgus             2       Northeast
                  unicirrhus                    Atlantic
                                                and South
                                                Africa

Collection                  Accession number
record

                   COl         COIII        rl6S

ENCB pending    KF225003     --           --

ENCB pending    KF225001     KF225007     KF373761

ENCB pending    KF225002     KF225008     --

ENCB pending    --           KF225009     KF373762

ENCB pending    KF225004     KF225010     KF373763

--              --           AJ250480     AJ390319
--              --           AJ012128     AJ390318
--              GU355923     GU355928     --
                  GU355926     GU355929
--              GU355924     GU355927     --
                  GU355925     GU355933
SBMNH           KF225005     KF225011     KF373764
  pending

SBMNH           KF225006     KF225012     KF373765
  pending
--              --           AJ250483     AJ390322

--              --           AJ012123     AJ390315

--              HQ214117     Pending *    Pending *

--              --           AJ616312     AJ616308

--              --           AJ250478     AJ390316
--              FN424379     --           --
--              AF377961     AJ250486     AJ414702

--              HM104263     AJ012129     AJ390324

-, the sequence is not available for the gene and there is
no voucher specimen; ENCB, Escuela Nacional de Ciencias
Biologicas; SBMNH, Santa Barbara Museum of Natural History.
* Flores-Valle et al. (unpubl.).


APPENDIX B

Topologies of Octopus hubbsorum-Octopus mimus specimens constructed using Bayesian analysis based on 3 genes: COI, COIII, and r16S. The values of the nodes indicate the probability posterior.

APPENDIX C

Topologies of Octopus hubbsorum-Octopus mimus specimens constructed using parsimony based on 3 genes: COI, COIII, and r16S. The bootstraps values are indicated below the nodes.

ACKNOWLEDGMENTS

We thank Ruben Garcia and Miguel A. Regalado for the Mexican octopus specimens donated from Oaxaca and Nayarit, respectively; and Alejandro Flores-Valle for allowing us to use unpublished sequences of Octopus maya. R. P. C. thanks the Consejo Nacional de Ciencia y Tecnologia (CONACYT) for the scholarship received. This study was supported by grant no. 147.09.01 to IDLABS

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RICARDO PLIEGO-CARDENAS, (1,2) * FREDERICK G. HOCHBERG, (3) FRANCISCO JAVIER GARCIA DE LEON (4,5) AND IRENE DE LOS ANGELES BARRIGA-SOSA (2)

(1) Doctorado en Ciencias Biologicas y de la Salud, Division CBS., Universidad Autonoma Metropolitana Unidad Iztapalapa, Av. San Rafael Atlixco 186, Col. Vicentina, Del. Iztapalapa, Mexico, Distrito Federal, 09340. Mexico; (2) Laboratorio de Genetica y Biologia Molecular, Planta Experimental de Production Acuicola, Departamento de Hidrobiologia, Universidad Autonoma Metropolitana Unidad Iztapalapa, Av. San Rafael Atlixco 186, Col. Vicentina, Del. Iztapalapa, Mexico, Distrito Federal, 09340. Mexico; (3) Department of Invertebrate Zoology, Santa Barbara Museum of Natural History, Santa Barbara, CA; (4) Laboratorio de Genetica Para la Conservation, Centro de lnvestigaciones Biologicas del Noroeste, S.C., La Paz, Baja California Sur 23090, Mexico; (5) Laboratorio de Ecologia Molecular, Fundacion CEQUA, c/21 de Mayo 1690 (esq. Bellavista), Punta Arenas, Chile

* Corresponding author. E-mail: rip_rojo@hotmail.com

DOI: 10.2983/035.033.0128

TABLE 1.
Primer reference and the annealing temperature of the primers
used in this study.

Gene    Annealing              Reference
       temperature
       ([degrees]C)

COI         49        Folmer et al. (1994)
com         38        Barriga-Sosa et al. (1995)
r16S        52        Simon et al. (1991)

TABLE 2.
Base composition per codon position for COI and COIII
partial sequences.

Species                       1st

                 T      C      A      G

Octopus         26.1   21.6   25.6   26.7
  mimus
Octopus         26.1   20.9   27.0   26.1
  hubbsorum
0. mimus        34.0   18.9   29.2   17.9
0. hubbsorum    33.9   19.4   28.3   18.4

Species                      2nd

                 T      C      A      G

Octopus         43.2   24.9   14.7   17.3
  mimus
Octopus         43.2   24.0   15.7   17.0
  hubbsorum
0. mimus        42.2   23.2   19.9   14.7
0. hubbsorum    41.3   23.3   20.5   14.9

Species                      3rd

                 T      C      A      G

Octopus         43.5   9.3    45.9   1.3     COI
  mimus
Octopus         44.0   9.3    45.9   0.9
  hubbsorum
0. mimus        45.9   12.0   41.1   0.9    COIII
0. hubbsorum    46.0   11.4   41.2   1.3

TABLE 3.
Variable nucleotide positions of partial sequences of COI
and COIII found between Octopus hubbsomm and Octopus mimus
haplotypes.

                                   COI Sites

Haolotypes   135   231   243   264   348   402   429   475
OhGC         C     A     C     C     A     T     T     A
OhNl         T
OhM          --    --    --    --    --    C     --    --
OhO          --    --    --    --    --    --    C     --
OmChl        --    G     T     T     G     --    --    G
OmCh2        --    G     --    T     G     --    --    G

                                   COIII Sites

Haplotypes   21    54    120   147   219   243   304   309
OhGC         A     C     C     T     T     C     T     T
OhNl         --    --    --    C     --    --    --    --
OhN2         T     --    --    --    --    --    --    --
OhM          --    --    T           --    a*    --    C
OmCR         --    --    --    C
OmChl        --    T     --    --    --    T     C     c
OmCh2        --    T     --    --    --    T     C     c
OmCh3        --    T     --    --    G     T     c     c

                       COIII Sites

Haplotypes   409   427   429   450   502
OhGC         G     A     C     T     G
OhNl         --    --    --    --    --
OhN2         --    --    --    C     --
OhM          --    --    --    --    --
OmCR
OmChl        --    G     A     --    A
OmCh2        A     G     A     --    A
OmCh3        --    G     A     --    --

OhGC, O. hubbsorum Gulf of California; OhM, O. hubbsorum
Michoacan; OhNl, O. hubbsomm Nayarit 1; OhN2, O. hubbsomm
Nayarit 2; OhO--O. hubbsorum Oaxaca; OmChl, O. mimus Chile
1; OmCh2, O. mimus Chile 2; OmCh3, O. mimus Chile 3; OmCR,
O. mimus Costa Rica.

TABLE 4.
Variable nucleotide positions of partial sequences of rl6S
found between Octopus hubbsorum and Octopus mimus
haplotypes.

r16S Sites

Haplotvpes   33   238   249   266   317   331   341   345
OhGC         A    G     C     A     G     G     T     A
OhN3         G    --    --    --    --    --    --    --
OhM          --   --    --    --    --    A     --    --
OmCR         --   --    --    --    --    --    --    --
OmCh3        --   A     T     G     A     --    C     G

OhGC, O. hubbsorum Gulf of California; OhM, O. hubbsorum
Michoacan; OhN3, O. hubbsorum Nayarit 3; OmCh,3 O. mimus
Chile 3; OmCR. O. mimus Costa Rica.

TABLE 5.
Genetic distances (percentage) calculated with the Tamura-
Nei model (Tamura & Nei 1993) based on mitochondrial
sequences of COI (first row), COIII (second row), and rl6S
(third row) for Octopus hubbsorum, Octopus maya and
Octopus mimus.

Taxa      OmCh(I)   OmCh(C)   OmP   OmCR   OspC   OhGC

OmCh(C)   --
          0.6
OmP       --        0.2
          0.4       0.2
OmCR      --        --        --
          1.4       1.6       1.4
          1.3       --        --
OspC      --        0.6       0.8   --
          1.2       1.4       1.2   0.2
          --        --        --    0.0
OhGC      --        0.8       0.9   --     0.2
          1.2       1.4       1.2   0.2    0.0
          1.3       --        --    0.0    0.0
OmyGM     --        9.2       8.9   --     9.0    9.0
          8.0       8.3       8.0   7.0    7.3    7.3
          4.5       --        --    5.5    5.5    5.5

-, missing sequences for the respective gene; OhGC, Octopus
hubbsorum Gulf of California; OmCh(C), Octopus mimus Coloso,
Chile; OmCh(I), O. mimus Iquique, Chile; OmCR, O. mimus Costa
Rica, OmP, O. mimus Peru; OmyGM, Octopus maya Gulf of Mexico;
OspC, Octopus sp. Colombia.
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Author:Pliego-Cardenas, Ricardo; Hochberg, Frederick G.; De Leon, Francisco Javier Garcia; Barriga-Sosa, Ir
Publication:Journal of Shellfish Research
Article Type:Report
Geographic Code:3CHIL
Date:Apr 1, 2014
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