DNA Barcoding revealing the occurrence of Isarachnanthus (Cnidaria; Anthozoa; Ceriantharia) in Cape Verde

. The occurrence of Isarachnanthus Carlgren, 1924 (Cnidaria: Anthozoa: Ceriantharia) specimens in Cape Verde Islands is recorded. Identification of the tube anemone species Isarachnanthus maderensis (Johnson, 1861) was possible based on DNA Barcoding. A discussion on biogeographic patterns associated with ocean circulation and life cycle is presented.


INTRODUCTION
The genera Arachnanthus Carlgren, 1912 andIsarachnanthus Carlgren, 1924 (Cnidaria: Anthozoa: Ceriantharia) correspond to the only ones with described adult forms of the family Arachnactidae (den Hartog, 1977;Stampar et al., 2016). This family is distinguished from the family Cerianthidae (most of the currently valid species; cf. Stampar et al., 2016) mostly due to the presence of planktonic larvae with long prevalence in the water column (Stampar et al., 2015). This peculiarity of the life cycle resulted in complex biogeographic patterns, since the dispersion based on this larval type seems to be quite effective (Stampar et al., 2012(Stampar et al., , 2015. Recently, Stampar et al. (2012) demonstrated that the distribution pattern for the genus Isarachnanthus in the Atlantic maintains the gene flow due to marine currents. The finding of specimens at the Ascension Island  demonstrated that the scenario proposed seems correct. However, the data available for the Eastern Atlantic records come from very distant areas, Madeira Island and Ascension Island (more than 4,500 km away). Thus, records in between those extreme regions are quite important to further test the scenario proposed. Consequently, this study presents occurrence data of the genus at the Cape Verde islands and discuss the relevance of biogeographic patterns based on life cycle aspects.

MATERIAL AND METHODS
Two specimens of Isarachnanthus ( Fig. 1) were sampled by SCUBA diving during night time in Santo Antão Island, Porto Novo harbour, nine meters depth, 11 November 2014 (1) and Tarrafal, Santiago Island, 27 October 2015 (2). Each animal was directly preserved in Ethanol 95%. These specimens were studied following the methods described by Stampar et al. (2012Stampar et al. ( , 2014 and Rach et al. (2017). The barcoding region (COI) was sequenced using Folmer et al. (1994) primers (619 bp after assembly) (GenBank MK904564 and MK904565) and then compared with data of Stampar et al. (2012) and . The voucher is deposited at Museu de Zoologia (USP) -MZUSP (MZSP 8468). P-distance model of base substitution was used to calculate genetic distances in MEGA7 software (Kumar et al., 2016). The maximum likelihood phylogenetic analysis was conducted via MEGA7 (500 replicates) with general time reversible model (GTR) (Kumar et al., 2016).

RESULTS
The sequences obtained were compared with the dataset available for the genus (after Stampar et al., 2012;. The first  Stampar, S.N. et al.: Ocurrence of Isarachnanthus (Ceriantharia) in Cape Verde Pap. Avulsos Zool., 2019; v.59: e20195940 2/4 approach was a phylogenetic reconstruction by maximum likelihood, which indicated two monophyletic clades (Fig. 2). The specimens obtained from Cape Verde were clustered among the specimens delimited in the clade of the species Isarachnanthus maderensis (Johnson, 1861). The estimated genetic distance (p-value) indicates that there is no variation in the DNA Barcoding sector between the Cape Verde, Madeira and Curaçao specimens (Table 1). At the same time, the variation for the Ascension Island specimens was not constant, for one specimen there was practically no variation, but for the other the variation was almost 8%. The distance to the other species, Isarachnanthus nocturnus (Hartog, 1977), was always constant between 7 and 8%. At the end, the DNA Barcode results indicate that the Cape Verdean specimens are Isarachnanthus maderensis.

This first record of the genus Isarachnanthus in Cape
Verde based on molecular data is very relevant because it presents information from an area closer to the coast of the African continent.  inferred that the coast line of the African continent should be inhabited only by the species Isarachnanthus maderensis, if the proposed evolutionary scenario is correct (Stampar et al., 2012). In this way, the present data maintain the 2012's proposal valid and still throw some light on the possible reproductive dynamics in this region. Some records have indicated the occurrence of Isarachnanthus nocturnus in the Eastern Atlantic (e.g., Bianchi et al., 2000), but results presented in this study and in  indicate that this occurrence is not likely -further investigation on these specimens should be conducted both morphological and molecularly.
The distribution of the studied specimens from Cape Verde, Madeira and Curaçao (Fig. 3) are under the influence of the North Atlantic Gyre currents and this can be demonstrated by the absence of genetic distance between these individuals (Table 1). This same pattern can be recognized in the other species of Isarachnanthus on the coast of South America and the Caribbean, where the specimens are under the influence of coastal currents from Argentina to the Caribbean Sea (Stampar et al., 2012). Both scenarios are maintained by the presence of a long-lived planktonic larva (Stampar et al., 2015) and the gene flow resulting from the action of these larvae is still very little known. Recently, an enormous phenotypic plasticity of the larvae has been discovered , which can also have influence on these patterns. Still in this approach, it is possible to recognize a distinct genetic pattern in relation to the studied specimens of Isarachnanthus maderensis from Ascension Island and Rocas Atoll (Table 1). This different pattern must be the result of a partial biogeographic isolation, since these two areas are mainly under influence of the South Atlantic Gyre currents. The influence of marine currents on genetic structuring in the Atlantic Ocean is already known, including fairly similar areas and patterns (e.g., Muss et al., 2001). However, the possible greater isolation of the population of the Ascension Island deserves better attention, because at some level this can result in complete isolation (and eventually leading to speciation) in future.