Submitted 7 December 2018Accepted 14 June 2019Published 29 July 2019

Corresponding authorElena A. Ritschard,ea.ritschard10@uniandes.edu.co

Academic editorJames Reimer

Additional Information andDeclarations can be found onpage 11

DOI 10.7717/peerj.7300

Copyright2019 Ritschard et al.

Distributed underCreative Commons CC-BY 4.0

OPEN ACCESS

First molecular approach to the octopusfauna from the southern CaribbeanElena A. Ritschard1,2, Jürgen Guerrero-Kommritz3 and Juan A. Sanchez1

1 Laboratorio de Biología Molecular Marina (BIOMMAR), Departamento de Ciencias Biológicas- Facultad deCiencias, Universidad de Los Andes, Bogotá, Colombia

2Department of Molecular Evolution and Development, University of Vienna, Vienna, Austria3 Fundación Fundabas, Bogotá, Colombia

ABSTRACTThe octopus fauna from the southern Caribbean is an understudied field. However,recent taxonomic work in the Colombian Caribbean has led to the discovery ofseveral new species in the family Octopodidae. To provide molecular evidence forrecent descriptions in the area (i.e., Octopus taganga, O. tayrona and Macrotritopusbeatrixi) and contribute to the systematics of the family, we reconstructed the firstmolecular phylogenies of the family including Colombian Caribbean octopus species.Using cytochrome c oxidase subunit I and rhodopsin sequences from specimens collectedin three sites (Santa Marta, Old Providence and San Andrés Islands) we inferredmaximum-likelihood trees and delimited species with PTP. Ourmitochondrial analysissupported the monophyly of species found in the area (i.e.,O. taganga,O. hummelinckiand O. briareus). The genetic distinction of the species O. tayrona and O. insularis wasnot resolved, as these were found in one clade together with Caribbean O. vulgarisand O. aff. tayrona species (O. spB) and delimited as a single species. Additionally,our results suggest a distant relationship of the Type I O. vulgaris group (Caribbeanregion) from the other forms of the species complex (Old World and Brazil). Lastly,the third newly described species M. beatrixi emerged as an independent lineage andwas delimited as a single species. However, its relationship to other species of its genusremains unknown due to the lack of sequences in databases. Altogether, our molecularapproach to the octopus fauna from the southern Caribbean adds on information to therelationship of Octopodidae species world-wide by providing sequences from recentlydescribed species from an understudied region. Further studies employing higher taxonsampling andmoremolecular information are needed to fill taxonomic gaps in the areaand account for single-locus resolution on the systematics of this group.

Subjects Biodiversity, Genetics, Molecular Biology, TaxonomyKeywords Octopodidae, Molecular phylogeny, Rhodopsin, Southern Caribbean, Speciesdelimitation, Octopus, Cytochrome c oxidase subunit I

INTRODUCTIONThe Caribbean region represents a global-scale hotspot of marine biodiversity, whichincludes the greatest concentration of marine species in the tropical Atlantic Ocean (Robertset al., 2002). However, many gaps in biodiversity studies remain and need to be filled inorder to define conservation priorities and design regional-scale management strategies(Miloslavich et al., 2010). An accurate evaluation of marine biodiversity in the Caribbean

How to cite this article Ritschard EA, Guerrero-Kommritz J, Sanchez JA. 2019. First molecular approach to the octopus fauna from thesouthern Caribbean. PeerJ 7:e7300 http://doi.org/10.7717/peerj.7300

needs comprehensive identification guides and taxonomic expertise. This phenomenonhas also been called taxonomic impediment (see Wheeler, 2004; Wheeler, Raven & Wilson,2004; Crisci, 2006) and, together with poor sampling efforts, represents an enduring issuefor many invertebrates (e.g., Coleman, 2015), including cephalopods (Norman & Hochberg,2005).

All of the major cephalopod lineages contain taxa with unstable systematics (Allcock,Strugnell & Johnson, 2008). In 2005, Norman and Hochberg reported 186 resolved octopusspecies names in the family Octopodidae d’Orbigny. Until then, around 50% of thedescribed species were lumped in the genus Octopus Cuvier, 1798 (Allcock, Strugnell &Johnson, 2008). This genus, sensu stricto, has only 11 accepted species, all other belong todifferent genera and most still need to be defined (Norman, Finn & Hochberg, 2014). Thishas led to an underestimation of several genera within Octopodidae. Regional surveysaround the globe additionally revealed a higher octopod diversity, with a recent increasein discovery of new species (Norman & Hochberg, 2005). Moreover, in recent years, studiesof cryptic species complexes have increased the number of putative species in the family(e.g., Amor et al., 2016; Amor et al., 2014; Leite et al., 2008). Despite these improvements tooctopod systematics, some regions around the globe remain understudied. For instance,within the highly diverse Caribbean, its southern area (Panama, Venezuela and Colombia)represents a remarkable taxonomic gap of theOctopodidae (Guerrero-Kommritz & Camelo-Guarin, 2016).

Twenty-nine (29) cephalopod species have been reported from theColombianCaribbean(Díaz, Ardila & Gracia, 2002; Díaz, Ardila & Gracia, 2000; Guerrero-Kommritz & Camelo-Guarin, 2016; Guerrero-Kommritz et al., 2016) and 12 of them belong to the familyOctopodidae sensu Strugnell et al. (2014). Recent taxonomic work based on morphology(Guerrero-Kommritz & Camelo-Guarin, 2016; Guerrero-Kommritz & Camelo-Guarin,2016; Guerrero-Kommritz & Rodriguez-Bermudez, 2018) from the Taganga Bay at SantaMarta, Colombia, revealed three newly described octopus species (i.e., Octopus tayronaGuerrero-Kommritz & Camelo-Guarin, 2016; O. taganga Guerrero-Kommritz & Camelo-Guarin, 2016, andMacrotritopus beatrixi Guerrero-Kommritz & Rodriguez-Bermudez, 2018)and at least seven more remain undescribed (J G-K, pers. obs., 2018), which highlightsthe currently unrecognized biodiversity of this understudied region. These morphologicalstudies performed in the country are the first step to understand the diversity of Colombianoctopod species, and molecular phylogenetic analyses comprise a next step to follow.Therefore, the purpose of this study was to assess the biodiversity of benthic shallow-wateroctopuses from three locations in the Colombian Caribbean using genetics by constructingthe first molecular phylogeny of the family Octopodidae that included Colombian samples.Particular goals were to: (1) providemolecular evidence for recently described species in thearea (namely,O. tayrona,O. taganga andM. beatrixi); and (2) contribute to the systematicsof the Octopodidae family by adding data from an understudied area of the globe.

Ritschard et al. (2019), PeerJ, DOI 10.7717/peerj.7300 2/14

Figure 1 Sampling locations at the Colombian Caribbean Sea. San Andrés Island (A), Old ProvidenceIsland (B) and Taganga Bay at Santa Marta (C). Maps were generated with QGIS.

Full-size DOI: 10.7717/peerj.7300/fig-1

MATERIALS AND METHODSSpecimensA total of 38 tissue samples and 36 specimens were obtained between February and Marchof 2016 with the help of local fishermen by hand capture with aid of a hook or a shovel(Guerrero-Kommritz & Camelo-Guarin, 2016; Guerrero-Kommritz & Rodriguez-Bermudez,2018). The sampling was performed in three areas with depths less than 7 m near the shoreat the Caribbean Sea: San Andrés Island, Old Providence Island and Taganga Bay at SantaMarta, Colombia (Fig. 1). Mantle tissues were stored in 90% ethanol at−20 ◦C until DNAextraction. Voucher specimens were fixed in formalin 4% for 24 to 48 h, preserved inethanol 70% and identified by morphometries and counts following Guerrero-Kommritzet al. (2016) catalogue for southern Caribbean octopod species. These were depositedin the ANDES Natural History Museum at Los Andes University, Bogota, Colombia(Table S1). The samples corresponded by morphological identifications (summarized inTable S2) to four genera and six described species. Namely,O. tayrona (Guerrero-Kommritz& Camelo-Guarin, 2016), O. taganga Guerrero-Kommritz & Camelo-Guarin, 2016, O.briareus Robson, 1929, O. hummelincki Adam, 1936, Amphioctopus burryi Voss, 1950and M. beatrixi (Guerrero-Kommritz & Rodriguez-Bermudez, 2018). Specimens belongingto the O. aff. tayrona complex (O. spB and O. spD; Guerrero-Kommritz & Camelo-Guarin,2016), which are currently being described as new species by J. G-K, and to the genusCallistoctopus Taki, 1964 were also found.

Ritschard et al. (2019), PeerJ, DOI 10.7717/peerj.7300 3/14

Research and collection were approved by the National Environmental LicensingAuthority (ANLA, Spanish acronym): Collection Framework Agreement granted toUniversidad de los Andes through resolution 1177 of October 9th, 2014 - IBD 0359.

Molecular techniquesDNA was obtained from tissues by phenol-chloroform extraction and ethanolprecipitation following the Coffroth et al. (1992) protocol. Genomic DNA was storedin ultrapure water at −20 ◦C until polymerase chain reaction (PCR) amplification.The mitochondrial gene cytochrome c oxidase subunit I (COI) was amplified usingthe primers LCO1490 and HC02198 (Folmer et al., 1994) and the nuclear geneRhodopsin (Rho) with the primers 5′-TTGCCTGTACTGTTTGCTAAAGC-3′ and 5′-GCCATCATTTCTTTCATTTGTGCGGCAT -3′(Strugnell, Norman & Drummond, 2004).

Each 15 µl PCR reaction contained the following reagents: 9.2 µl of ddH2O, 1.5 µl of10x buffer, 2.1 µl of 25 mM MgCl2, 0.3 µl of 10 mM dNTPs, 0.3 µl of 5 U/ µl Taq DNApolymerase (Promega), 0.3 µl of 10 mM of both the forward and reverse primers and 1 µlof 20 ng/ µl DNA. PCR thermal cycling program for COI was: denaturation at 92 ◦C for 5min; 35 cycles of 94 ◦C for 50 s, 54 ◦C for 1 min and 72 ◦C for 1 min; and final extension at72 ◦C for 7 min completed each PCR. Thermal cycling program for Rho was: denaturationat 94 ◦C for 2 min; 35 cycles of 94 ◦C for 40 s, 50 ◦C for 40 s and 72 ◦C for 90s; and finalextension at 72 ◦C for 10 min. PCR products were purified with the ExoSAP-IT protocol(Thermo Fisher Scientific) and sequenced by Macrogen Inc (Korea) in both directionsusing the same primers as for the PCR amplifications. Sequences generated in this studyare available under the GenBank accession numbers MG778037–MG778111 (Table S1).

Taxon samplingTo assess relationships between the individuals found in the three sampling sites andtheir position within the Octopodidae, the dataset included COI and Rho sequences fromdifferent sources (Table S3) of species of the family as the ingroup, and EnteroctopusdofleiniWülker, 1910 andMuusoctopus Gleadall, 2004 sequences as outgroup. The ingroupwas selected taking into account the identification to genus of the specimens collected,which belonged all to the family Octopodidae sensu Strugnell et al. (2014). The outgroupspecies were selected because these were found to be part of the Octopodidae sister group,following the latest Octobrachia phylogeny (Sanchez et al., 2018).

Phylogenetic analyses and species delimitationHaplotypes of the COI and Rho datasets were firstly collapsed using the online fastasequence toolbox FaBox 1.41 (http://www.birc.au.dk/software/fabox). Sequences werealigned with MAFFT v7 (multiple sequence alignment method based on fast Fouriertransform (FFT); Katoh & Standley, 2013) with default parameters. Alignments werecleaned with trimAl v1.4 (automated trimming alignment tool; Capella-Gutierrez, Silla-Martinez & Gabaldon, 2009) using a 0.5 gap threshold, 0.25 residue overlap and 90%sequence overlap to erase non-informative sites and sequences. The best-fit model foreach dataset was selected with ModelFinder (Kalyaanamoorthy et al., 2017) implementedin IQTree v1.6.2 (Nguyen et al., 2014) using the corrected Akaike Information criterion

Ritschard et al. (2019), PeerJ, DOI 10.7717/peerj.7300 4/14

(AICc). For both COI and Rho datasets, the Generalized Time-Reversible plus Gamma(GTR + G) model of nucleotide substitution was assigned. Best scoring maximum-likelihood trees were constructed with RAxML v8.2.11 (Randomized AxeleratedMaximumLikelihood; Stamatakis, 2014) using the rapid bootstrap algorithm, 1,000 bootstrapreplicates and 20 alternative runs on distinct starting trees. These resulting trees werefinally used as input for the species delimitation analysis with the maximum-likelihoodPoisson Tree Process method (PTP; Zhang et al., 2013) as implemented in the web server(https://species.h-its.org/ptp/). Analyses were performed for each gene separately andbootstrap supports >70% were considered significant. Final datasets (sequence files withunique haplotypes), alignments and tree files are available in (Data S1).

RESULTSThe COI (625 base pairs, 164 sequences) and Rho (235 base pairs, 40 sequences)maximum-likelihood trees of the family Octopodidae are depicted in Figs. 2 and 3, respectively. TheCOI tree recovered well-supported relationships (bootstrap/BS >70%) mainly at thespecies level (Fig. 2), whereas deeper branches remain unresolved (BS < 70%). The Rhotree (Fig. 3) included fewer sequences than the COI tree due to the limited availability ofthis molecular marker in GenBank, resulting in a lower representation of species from thefamily. Relationships in this analysis were found to be in general discrepant with the COItree, as detailed below. However, most of these topology inconsistencies were supportedbelow the 70% support level.

The maximum-likelihood approach with the mitochondrial gene (Fig. 2) recoveredthe monophyly of three species found in the studied area, namely O. hummelincki,O. briareus and O. taganga (BS = 100%). These were also delimited as single specieswith the PTP analysis (92%, 77% and 92% BS support, respectively; detailed PTP resultsin the Data S1). The recently described species M. beatrixi resulted as a sister lineage of awell-supported clade (BS = 85%) containing Wunderpus photogenicus and Thaumoctopusmimicus, although with a low bootstrap support (BS = 65%). The Callistoctopus sp.specimen (SM1) was found to be a sister lineage to the two C. ornatus representatives fromHawaii and Australia (BS = 85%) and the A. burryi sample (2017–01) was found withinits genus as a sister lineage to A. arenicola, A. aegina and A. marginatus (BS = 100%).These three Colombian samples (i.e., M. beatrixi, Callistoctopus SM1. and A. burryi) weredelimited each as single species with PTP (BS = 100%).

Conversely, the monophyly of the species O. tayrona could not be resolved. This isbecause O. vulgaris representatives of Puerto Rico and the Lesser Antilles (Caribbeanregion), and O. insularis (Leite et al., 2008) from Brazil and the mid- Atlantic Islandsappeared nested in the same clade as O. tayrona sequences with a bootstrap support of100% (Fig. 2). The undescribed speciesO. spD andO. spB proposed byGuerrero-Kommritz& Camelo-Guarin (2016) and O.s sp. samples from Old Providence Island resulted withinthis clade as well, in both mitochondrial and nuclear phylogenetic analyses (Figs. 2 and3). The PTP analysis with COI delimited this clade as one single species (BS = 89%;Fig. 2), whereas the Rho delimitation resulted in the delimitation of individual sequences

Ritschard et al. (2019), PeerJ, DOI 10.7717/peerj.7300 5/14

A. C.

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KF844044.1_Octopus_hummelincki_isolate_OhumCE1

MG778072.1_Macrotritopus_beatrixi_2017-02

MG778037.1_Octopus_hummelincki_isolate_2015−42

MG778073.1_Callistoctopus_sp._SM1

MG778074.1_Amphioctopus_burryi_isolate_2017−01

GQ900746.1_Thaumoctopus_mimicus_isolate_ThauMim11GQ900748.1_Wunderpus_photogenicus_isolate_Wunder24

HM104254.1_Abdopus_aculeatus

HM104260.1_Macroctopus_maorum

AY545188.1_Octopus_kaurna

AY616892.1_Callistoctopus_ornatus

HM104259.1_Grimpella_thaumastocheirHM104255.1_Ameloctopus_litoralis

GQ900730.1_Amphioctopus_marginatus_isolate_AmphMarg29

AF000043.1_Hapalochlaena_maculosa

KC894940.1_Octopus_salutii_isolate_OSA2KC894941.1_Octopus_salutii_isolate_OSA4

GQ900732.1_Callistoctopus_ornatus_isolate_CallOrna85

GQ900731.1_Callistoctopus_luteus_isolate_CallLute77

GQ900729.1_Amphioctopus_arenicola_isolate_AmphAren74

AB430535.1_Octopus_cyaneaAB191280.1_Octopus_cyaneaAB430534.1_Octopus_cyanea

AB430544.1_Octopus_parvusHM104258.1_Cistopus_indicus

AB430542.1_Octopus_incellaAB191271.1_Octopus_parvus

AB430545.1_Octopus_wolfi

AB191275.1_Callistoctopus_minorAB191277.1_Octopus_sasakii

HQ846124.1_Amphioctopus_kagoshimensis_isolate_OK3

HQ846140.1_Amphioctopus_marginatus_isolate_TS3AB191276.1_Amphioctopus_aeginaHQ846138.1_Amphioctopus_marginatus_isolate_TS1

HQ846158.1_Amphioctopus_ovulum_isolate_SP45HQ846159.1_Amphioctopus_ovulum_isolate_SP47HQ846157.1_Amphioctopus_ovulum_isolate_SP44

HQ846155.1_Amphioctopus_fangsiao_isolate_L21HQ846163.1_Hapalochlaena_maculosa_isolate_R4

AB430543.1_Octopus_laqueus

HM104263.1_Scaeurgus_unicirrhus

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KF844003.1_Octopus_insularis_isolate_OinCE38

KF844000.1_Octopus_insularis_isolate_OinPA14

KF844010.1_Octopus_insularis_isolate_OinRN12KF844016.1_Octopus_insularis_isolate_OinRN34

KF844020.1_Octopus_insularis_isolate_OinPE1KF844025.1_Octopus_insularis_isolate_OinBA199KF844022.1_Octopus_insularis_isolate_OinBA05

KF844024.1_Octopus_insularis_isolate_OinBA125

KF844018.1_Octopus_insularis_isolate_OinPB266

KF844030.1_Octopus_vulgaris_isolate_OvuPA184KF844031.1_Octopus_vulgaris_isolate_OvuBA117KF844040.1_Octopus_vulgaris_isolate_OvuSC10KF844026.1_Octopus_vulgaris_isolate_OvuAP225

KF844027.1_Octopus_vulgaris_isolate_OvuPA173

KT335828.1_Octopus_bimaculatus_isolate_83

AY545189.1_Octopus_bimaculoides

KF225005.1_Octopus_hubbsorum_isolate_Col1

MG778048.1_Octopus_tayrona_isolate_SM18

MG778055.1_Octopus_tayrona_isolate_SA8

MH550425.1_Octopus_insularis_isolate_P1IE

JX500652.1_Octopus_vulgaris_voucher_MMIIM_35MG778042.1_Octopus_sp._PR3JX500654.1_Octopus_vulgaris_voucher_MMIIM_37

JX500622.1_Octopus_vulgaris_voucher_MMIIM_5JX500651.1_Octopus_vulgaris_voucher_MMIIM_34MG778056.1_Octopus_tayrona_isolate_SM16MH550423.1_Octopus_insularis_isolate_P3AA

MG778041.1_Octopus_sp._B_PR2

MG778051.1_Octopus_tayrona_isolate_SM21

MG778038.1_Octopus_tayrona_isolate_SM2MH550422.1_Octopus_insularis_isolate_P2AA

MG778040.1_Octopus_sp._PR1

MG778047.1_Octopus_tayrona_isolate_SM17MG778050.1_Octopus_tayrona_isolate_SM20MG778043.1_Octopus_sp._PR4

MG778044.1_Octopus_sp._PR5

JX500649.1_Octopus_vulgaris_voucher_MMIIM_32

GU362545.1_Octopus_maya

MG778063.1_Octopus_taganga_isolate_SM7MG778065.1_Octopus_taganga_isolate_SM11

MG778066.1_Octopus_briareus_isolate_SA1

MG778070.1_Octopus_sp._SA6JX500618.1_Octopus_briareus_voucher_MMIIM_1

MG778069.1_Octopus_briareus_isolate_SA5

MG778071.1_Octopus_briareus_isolate_SA4

KF489451.1_Octopus_vulgaris

KJ605276.1_Octopus_cf._tetricus_WAM6708

KJ605264.1_Octopus_cf._tetricus_SWA006KJ605268.1_Octopus_cf._tetricus_SWA010KJ605269.1_Octopus_cf._tetricus_WAM6701KJ605277.1_Octopus_cf._tetricus_WAM6709KJ605270.1_Octopus_cf._tetricus_WAM6702

KJ605262.1_Octopus_cf._tetricus_CT123KJ605263.1_Octopus_cf._tetricus_CT133

KJ605247.1_Octopus_tetricus_isolate_NQ002KJ605251.1_Octopus_tetricus_isolate_NQ015KJ605261.1_Octopus_tetricus_isolate_NZ1KJ605254.1_Octopus_tetricus_isolate_TAS4113KJ605258.1_Octopus_tetricus_isolate_LML1KJ605256.1_Octopus_tetricus_isolate_TAS4124KJ605252.1_Octopus_tetricus_isolate_NQ028KJ605250.1_Octopus_tetricus_isolate_NQ011KJ605249.1_Octopus_tetricus_isolate_NQ010KJ605248.1_Octopus_tetricus_isolate_NQ003KJ605253.1_Octopus_tetricus_isolate_NQ029KU525760.1_Octopus_jollyorum_isolate_EBU54865

HQ908434.1_Octopus_vulgaris_voucher_OVMed09HQ908435.1_Octopus_vulgaris_voucher_OVMed10

HQ908433.1_Octopus_vulgaris_voucher_OVMed08HQ908436.1_Octopus_vulgaris_voucher_OVMed11

KJ605284.1_Octopus_vulgaris_isolate_OVAL5KJ605280.1_Octopus_vulgaris_isolate_OVAL1KJ605282.1_Octopus_vulgaris_isolate_OVAL3

JX500626.1_Octopus_vulgaris_voucher_MMIIM_9

JX500629.1_Octopus_vulgaris_voucher_MMIIM_12JX500636.1_Octopus_vulgaris_voucher_MMIIM_19

JX500632.1_Octopus_vulgaris_voucher_MMIIM_15

JX500630.1_Octopus_vulgaris_voucher_MMIIM_13JX500633.1_Octopus_vulgaris_voucher_MMIIM_16HQ908429.1_Octopus_vulgaris_voucher_OVMed04

HQ908426.1_Octopus_vulgaris_voucher_OVMed01HQ908428.1_Octopus_vulgaris_voucher_OVMed03HQ908432.1_Octopus_vulgaris_voucher_OVMed07HQ908431.1_Octopus_vulgaris_voucher_OVMed06

HQ908427.1_Octopus_vulgaris_voucher_OVMed02HQ908430.1_Octopus_vulgaris_voucher_OVMed05

JX500631.1_Octopus_vulgaris_voucher_MMIIM_14

KJ605283.1_Octopus_vulgaris_isolate_OVAL4

DQ683209.1_Octopus_vulgaris_isolate_OHB4DQ683211.1_Octopus_vulgaris_isolate_OSB6

DQ683214.1_Octopus_vulgaris_isolate_ODB1DQ683216.1_Octopus_vulgaris_isolate_ODB3

KJ605279.1_Octopus_vulgaris_isolate_SAVULG01

KP056552.1_Octopus_insularis_isolate_Q5901

DQ683205.1_Octopus_vulgaris_isolate_OTC2

FN424379.1_Octopus_vulgaris

KU525758.1_Octopus_jollyorum_isolate_MA11TaiAB430546.1_Octopus_sinensisHQ846154.1_Octopus_sinensis_isolate_K3

AB430532.1_Octopus_oliveri

JX500655.1_Octopus_vulgaris_voucher_MMIIM_38

JX500664.1_Octopus_vulgaris_voucher_MMIIM_47KU525769.1_Octopus_vulgaris_isolate_OvMo1

JX500666.1_Octopus_vulgaris_voucher_MMIIM_49JX500676.1_Octopus_vulgaris_voucher_MMIIM_59JX500677.1_Octopus_vulgaris_voucher_MMIIM_60KF844042.1_Octopus_vulgaris_isolate_OvuPT5DQ683221.1_Octopus_vulgaris_isolate_OGA1

KU525761.1_Octopus_vulgaris_isolate_OvMO2JX500660.1_Octopus_vulgaris_voucher_MMIIM_43

KF774309.1_Octopus_bimaculoides

AF377967.1_Octopus_bimaculoides

KF225001.1_Octopus_hubbsorum_isolate_M5KF225003.1_Octopus_hubbsorum_isolate_Oax3KF225004.1_Octopus_hubbsorum_isolate_GC90

KP056551.1_Octopus_mimus_isolate_mimusPa3GU355924.1_Octopus_mimus_isolate_L25I

GU355925.1_Octopus_mimus_isolate_L31I

GU355923.1_Octopus_mimus_isolate_C10IKP056550.1_Octopus_mimus_isolate_mimusM2

KU525767.1_Octopus_vulgaris_isolate_MA10Chl

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O. vulgaris type I (?)

O. insularis

O. tayrona

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O. briareus

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O. vulgaris type III

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O. sinensis

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Figure 2 Cytochrome c oxidase subunit I (COI) Maximum Likelihood tree. (B–C) Black dots representbootstrap support over the 70% threshold, and asterisks (*) demark sequences generated in this study.Thick grey bars to the right of the phylogeny represent the delimitation of species using PTP with a boot-strap support higher than 70%. Clades with no such bars were not resolved by the species delimitationanalysis. Sequences are colored corresponding to their area of sampling (map in A). Abbreviations as fol-low - mPac: mid-Pacific; nePac: north-eastern Pacific; sePac: south-eastern Pacific; Ca: Caribbean; Br:Brazil; mAI: mid-Atlantic Islands; SAf: South Africa; nAf: northern Africa; neA: north-eastern Atlantic;Me: Mediterranean Sea; mIO: mid-Indian Ocean Islands; Ind: India; IndPac: Indo-Pacific; nAs: northernAsia. The map template was downloaded from www.onlyGFX.com.

Full-size DOI: 10.7717/peerj.7300/fig-2

within this clade as single species (Fig. 3). Moreover, this highly-supported clade resultedin the COI tree (Fig. 2) as sister lineage of O. maya (BS = 45%), followed by O. taganga(BS = 70%). In the Rho tree the latter resulted to be paraphyletic to the O. aff tayronacomplex (Fig. 3), thus not establishing two separate groups. However, this relationshipwas supported below the 70% bootstrap level (55%). Other species relationships foundin the three sampling areas were found to be discordant between the COI and Rho tree.For instance, in the nuclear analysis (Fig. 3) O. hummelincki appeared nested within theO. tayrona clade (BS= 85%). Conversely, Callistoctopus SM1 was found, as in the COI tree,in a clade with C. ornatus and O. kaurna (BS = 80%; Fig. 3). Moreover, the PTP analysiswith the nuclear marker resulted in the delimitation of 10 species supported over the 70%bootstrap level (Fig. 3). However, many of these results were non-concordant with thephylogenetic signal (e.g., delimitation of sequences within the O. aff tayrona/O. taganga assingle species; Fig. 3).

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MG778109.1_Macrotritopus_beatrixi_2017−02_EAR−2018

MG778081.1_Octopus_tayrona_isolate_SM20

MG778088.1_Octopus_tayrona_isolate_SM18MG778089.1_Octopus_tayrona_isolate_SM17

MG778090.1_Octopus_tayrona_isolate_SM16

MG778098.1_Octopus_tayrona_isolate_SM10MG778103.1_Octopus_sp._PR5_EAR−2018

MG778100.1_Octopus_sp._B_PR2_EAR−2018

MG778097.1_Octopus_tayrona_isolate_SM9

MG778099.1_Octopus_sp._B_SM19_EAR−2018MG778078.1_Octopus_tayrona_isolate_SM3MG778086.1_Octopus_tayrona_isolate_SM6MG778076.1_Octopus_tayrona_isolate_SM21MG778075.1_Octopus_tayrona_isolate_SM2

MG778095.1_Octopus_tayrona_isolate_SM13MG778096.1_Octopus_tayrona_isolate_SM14MG778087.1_Octopus_sp._PR4_EAR−2018MG778093.1_Octopus_tayrona_isolate_SM8

MG778094.1_Octopus_tayrona_isolate_SM15

MG778092.1_Octopus_hummelincki_isolate_2015−42

MG778102.1_Octopus_taganga_isolate_SM11MG778110.1_Octopus_taganga_isolate_SM12MG778101.1_Octopus_taganga_isolate_SM7

AY545172.1_Octopus_bimaculoidesMG778108.1_Amphioctopus_burryi_isolate_2017−01

MG778107.1_Octopus_briareus_isolate_SA4MG778104.1_Octopus_briareus_isolate_SA5

MG778111.1_Callistoctopus_sp._SM1_EAR−2018AY545169.1_Octopus_kaurna

AY616926.1_Callistoctopus_ornatus

HM104287.1_Abdopus_aculeatus

AY545171.1_Hapalochlaena_maculosaHM104288.1_Ameloctopus_litoralis

AY545168.1_Octopus_berrima

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HM104297.1_Octopus_vulgaris

HM104291.1_Cistopus_indicus

HM104298.1_Scaeurgus_unicirrhus

AY545170.1_Octopus_rubescens

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Ca

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SAfmAI

mIO

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Figure 3 Rhodopsin (Rho) Maximum Likelihood tree. (B) Black dots represent bootstrap support overthe 70% threshold, and asterisks (*) demark sequences generated in this study. Thick grey bars to the rightof the phylogeny represent the delimitation of species using PTP with a bootstrap support higher than70%. Clades with no such bars were not resolved by the species delimitation analysis. Sequences are col-ored corresponding to their area of sampling (map in A). Abbreviations as follow - mPac: mid-Pacific;nePac: north-eastern Pacific; sePac: south-eastern Pacific; Ca: Caribbean; Br: Brazil; mAI: mid-Atlantic Is-lands; SAf: South Africa; nAf: northern Africa; neA: north-eastern Atlantic; Me: Mediterranean Sea; mIO:mid-Indian Ocean Islands; Ind: India; IndPac: Indo-Pacific; nAs: northern Asia. The map template wasdownloaded from www.onlyGFX.com.

Full-size DOI: 10.7717/peerj.7300/fig-3

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DISCUSSIONRegional findingsOur study provides, for the first time, molecular data from octopods found in theColombian Caribbean and newly described species from this area. The mitochondrialanalysis (Fig. 2) resolved the monophyly (BS = 100%) and species delimitation (BS= 92%) of O. taganga, the third ocellated species (with O. hummelincki and O. maya)from the Atlantic. Morphologically, O. taganga is closely related to O. hummelincki andappears to be related to the Pacific Ocean species Octopus oculifer (Hoyle, 1904), Octopusbimaculoides Pickford and McConnaughey, 1994 and Octopus bimaculatus Verrill, 1883(Guerrero-Kommritz & Camelo-Guarin, 2016). However, preliminary molecular resultsshowed it as more similar to O. maya than any other species present in the NCBI database(Guerrero-Kommritz & Camelo-Guarin, 2016). We found in the COI tree (BS = 70%)O. taganga as sister lineage of the O. tayrona/O. insularis clade and O. maya (Fig. 2). Thisgroup, together with O. mimus and O. hubbsorum, showed a close relationship with theocellated species O. bimaculoides and O. bimaculatus (BS = 100%).

The species O. tayrona formed a monophyletic group with O. insularis, CaribbeanO. vulgaris, Octopus sp. B, and Octopus sp. sequences from Old Providence Island in theCOI analysis (Fig. 2, BS = 100%) and was delimited as one single species with PTP (BS =89%). Until its description, O. tayrona and any other large octopus in the Caribbean wereidentified as O. vulgaris (Guerrero-Kommritz & Camelo-Guarin, 2016). However, it can bemorphologically distinguished from European O. vulgaris by its smaller ligula (O. tayrona:0.6 –1.6 mm; O. vulgaris: 1.2 –2.1 mm) and the absence of enlarged suckers in all arms,both in males and females. O. tayrona can be additionally distinguished from other similarspecies, such as O. insularis, mainly by its smaller size, the presence of large warts on thearms’ base and the absence of enlarged suckers on arms II and III (Guerrero-Kommritz &Camelo-Guarin, 2016). These features, together with other reproduction-related characters(e.g., relative size of the hectocotylus, presence and form of the calamus) and internalstructures related to digestion (e.g., size of liver, teeth on radula, form of salivary glandsand structure of stomach), are the base for morphological octopod species delimitation dueto their role on ecological adaptation, reproductive isolation and, consequently, speciation.Currently, O. tayrona is considered by morphology part of the O. vulgaris type I group(Guerrero-Kommritz & Camelo-Guarin, 2016), the species complex form representingthe wider Caribbean area (Norman, Finn & Hochberg, 2014). The inclusion of sequencesidentified as O. vulgaris from Puerto Rico and the Lesser Antilles within this clade in ourCOI tree either supports this claim or, most probably, indicates misidentification of thespecimens, as already suggested by Lima et al. (2017). Regardless, these phylogenetic resultscreate a conflict with themorphological species delimitation based on reproduction-relatedcharacters of O. tayrona and O. insularis. Moreover, the Rho tree consistently resulted ona highly supported (BS = 85%) monophyletic group of O. tayrona, O. sp. B and O. sp.from Old Providence. An O. insularis sequence from (González-Gómez et al., 2018) wasincluded in the analysis, but during trimming it was erased due to its low coverage inthe alignment. Therefore, together with the lack of rhodopsin sequences for Caribbean

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O. vulgaris, the mitochondrial results regarding O. tayrona could not be confirmed withthe nuclear marker. Future work using more molecular data would be necessary to followup these results. This would help to further evaluate the evolutionary relationship betweenO. tayrona, O. insularis and Type I O. vulgaris and confirm or reject their taxonomicaldistinction.

The newly described speciesMacrotritopus beatrixi resulted as an independent lineage inour analyses (Figs. 2 and 3) and was delimited as a single species (COI, BS = 100%; Rho,BS = 93%). So far, it represents the first published sequence of its genus. Thus, data fromotherMacrotritopus species are needed to resolve the genetic distinction of this new species.Moreover, our COI phylogeny and PTP results suggest that Callistoctopus SM1 from SantaMarta represents one independent lineage and a single species (BS = 100%), differentfrom all other representatives of this genus included in the analysis (Fig. 2). A new speciesof Callistoctopus is currently under description by J. G-K., expected to be closely relatedto C. macropus Risso, 1826 from the east Atlantic Ocean and Mediterranean Sea (due tomorphological similarities; J G-K, pers. obs., 2018). Callistoctopus macropus is also reportedfor the southern Caribbean (Díaz, Ardila & Gracia, 2000; Guerrero-Kommritz & Camelo-Guarin, 2016). However, after analyzing specimens from France the morphometrics andcounts were different, and we considered this a misidentification (research in process J.G-K). Unfortunately, the sequence generated here was taken from an individual whosewhole body could not be collected. Therefore, its morphological identification to specieslevel was not possible to achieve. Moreover, due to the lack of sequences of C. macropusin databases, their evolutionary relationship could not be molecularly assessed here andwarrants further investigation.

Contributions to the Octopodidae systematicsOur COI and Rho phylogenies help to fulfill the taxonomic gap in the Octopodidaefamily of species previously lacking molecular information and thus not consideredin the phylogenetic systematics of the family (i.e., O. tayrona, O taganga, A. burryiand M. beatrixi). For instance, the Macrotritopus beatrixi sample contributes the firstsequences of its genus, providing new insights on the Octopodidae genus-level systematics.Additionally, the inclusion of O. tayrona in our COI analysis, considered morphologicallyas part of the Type IO. vulgaris group, allowed the inference of the evolutionary relationshipof this species with other forms of the species complex. Our results suggest that O. tayronais more closely related to other species also found in the New World (e.g., O. taganga fromColombia, O. hubbsorum, O. bimaculatus and O. maya fromMexico, O. mimus from Chileand Peru, O. bimaculoides from California, USA) than to the O. vulgaris forms from theOld World and Brazil (Fig. 2). Moreover, its genetic distinction with O. insularis remainsunresolved.

Our mitochondrial analysis showed a better-resolved topology, with less polytomiesand more significantly-supported branches (BS > 70%) than the Rho tree. This couldbe explained by the general smaller effective population size of mitochondrial DNAcompared to nuclear genes (Moore, 1995). This leads to a higher probability of coalescencein short internodes and can result on a more accurate recovery of the species-tree topology.

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However, even awell-resolved gene tree can hardly resemble a species tree due to incompletelineage sorting and DNA barcodes are known to decrease their performance on poorlysampled groups (Meyer & Paulay, 2005). Moreover, recent genome-wide findings onthe O. vulgaris species complex (Amor et al., 2019) highlight that phylogenetic analysesusing only mitochondrial DNA underestimate octopus species diversity. Thus, despite thebetter performance of our COI analysis, more informative nuclear markers are needed tocomplement our results.

The number of cephalopod species in the Caribbean should increase as more surveysare made (Judkins et al., 2010; Miloslavich et al., 2010). Here we show a first attempt tofulfill taxonomic gaps on the octopod species of an understudied area of the highly diverseCaribbean. However, more sampling and taxonomic efforts are still needed to contribute tothe local and the world-wide picture of the Octopodidae systematics. Therefore, for futurestudies we suggest the inclusion of more taxa poorly represented in databases to accountfor incomplete taxon sampling and further tests using more molecular information toaccount for insufficient single-locus resolution.

CONCLUSIONSHere we present the first molecular approach to the octopus fauna from the southernCaribbean in Colombia. Our mitochondrial analysis supports the monophyly of speciesfound in the Colombian Caribbean, like Octopus taganga, O. hummelincki and O. briareus.The genetic distinction of the species O. insularis and O. tayrona was not resolved, asthese were found in one unresolved clade and delimited as one species together withCaribbean Octopus vulgaris and Octopus aff. tayrona species (O. spB). Moreover, ourresults suggest a distant relationship of the O. vulgaris-like species from the Caribbeanregion (e.g., O. tayrona and O. insularis) from the O. vulgaris species complex forms ofthe Old World and Brazil. Lastly, the newly described species Macrotritopus beatrixi wasdelimited as a single species but its relationship to other species of its genus remainsunknown. Altogether, our work contributes to the fulfilment of taxonomic gaps in theOctopodidae family and adds molecular evidence for recent descriptions in this area. Wesuggest for future studies to use more molecular markers to compensate for insufficientsingle-locus resolution. Furthermore, molecular data of poorly represented species shouldbe amended to databases to improve taxon sampling.

ACKNOWLEDGEMENTSSpecial thanks to the fishermen JuliánMatos (Taganga, Santa Marta), ‘‘Waily’’ (San AndrésIsland) and Casimiro Newball (Old Providence Island) for their help on the collectionof specimens; and to Carlos Pardo for his support on the data analysis. Thanks to theBiommar lab team for their support in laboratory procedures and discussion.

Ritschard et al. (2019), PeerJ, DOI 10.7717/peerj.7300 10/14

ADDITIONAL INFORMATION AND DECLARATIONS

FundingThis work was funded by Vicerrectoría de Investigaciones—Universidad de los Andes,under the research program ‘‘Completando la teoría del origen de las especies de Darwin:especiación ecológica en octocorales, desde niños a doctores’’. The funders had no rolein study design, data collection and analysis, decision to publish, or preparation of themanuscript.

Grant DisclosuresThe following grant information was disclosed by the authors:Vicerrectoría de Investigaciones—Universidad de los Andes.

Competing InterestsJürgen Guerrero-Kommritz is employed by Fundación Fundabas.

Author Contributions• Elena A. Ritschard conceived and designed the experiments, performed the experiments,analyzed the data, prepared figures and/or tables, authored or reviewed drafts of thepaper, approved the final draft.• Jürgen Guerrero-Kommritz and Juan A. Sanchez conceived and designed theexperiments, contributed reagents/materials/analysis tools, authored or reviewed draftsof the paper, approved the final draft.

Animal EthicsThe following information was supplied relating to ethical approvals (i.e., approving bodyand any reference numbers):

Research and collection of specimens were approved by National EnvironmentalLicensing Authority (ANLA, Spanish acronym): Collection Framework Agreement grantedto Universidad de los Andes through resolution 1177 of October 9th, 2014 - IBD 0359.

DNA DepositionThe following information was supplied regarding the deposition of DNA sequences:

The sequences generated here are accesible via GenBank accession numbers MG778037to MG778111. Sequences can be also found in Data S1.

Data AvailabilityThe following information was supplied regarding data availability:

Sequences and alignments used for the phylogenetic analyses and resulting trees forboth cytochrome c oxidase subunit I and rhodopsin genes are available in Data S1.

Supplemental InformationSupplemental information for this article can be found online at http://dx.doi.org/10.7717/peerj.7300#supplemental-information.

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