Color pa erns of the hermit crab Calcinus tibicen ( Herbst , 1791 ) fail to indicate high genetic variation within COI gene

Apart from traditional characters, other data have been used for taxonomy, like color patt erns. Based on the diff erent colors (green and orange) observed for some Calcinus tibicen (Herbst, 1761) specimens, we evaluated the genetic distance for cytochrome oxidase subunit I mitochondrial gene of individuals collected in Pernambuco (northern Brazil) and in São Paulo (southeast Brazil). We found low genetic variation (0.2–1.1%), and no evidence of isolation on our molecular tree based on genetic distance. We suggest high levels of gene fl ow between specimens with diff erent color patt erns, which are polymorphisms and might be related to the kind of nutrition as well diff erent ecological and evolutionary predation characteristics. keY woRds Anomura, COI gene, coloration, Diogenidae, polymorphism. sHoRt commUnIcAtIon This article is part of the special series offered by the Brazilian Crustacean Society in honor to Ludwig Buckup in recognition of his dedication and contributions to the development of Carcinology CORRESPONDING AUTHOR Fernando Luis Mantelatto mantel@usp.br SUBMITTED 28 August 2017 ACCEPTED 22 November 2017 PUBLISHED 26 March 2018 Guest Editors Alessandra A. de Pádua Bueno and Sandro Santos DOI 10.1590/2358-2936e2018008 orcid.org/0000-0002-3092-9588

Low genetic variation in colored specimens of C. tibicen Nauplius, 26: e2018008 Morphological characters have been used as the main tool of identification and description of decapod crustaceans, but other information such as life history and development, habitat, mating behavior, coloration, and molecular data have become useful for species recognition (Knowlton, 1986;Malay and Paulay, 2009).With the improvement of optical photo equipment and more facilities during field collections during the last two decades, taxonomists have used more frequently color patterns of living animals or the residual patterns found in preserved specimens for species-specific recognition or identification in some decapod groups (e.g.Salmon et al., 1979;Williams and Felder, 1986;Bruce, 1978;Malay and Paulay, 2009;Negri et al., 2014;Hamasaki et al., 2017).
Among hermit crabs, color pattern differentiation is quite significant between cryptic species (e.g.Asakura and Paulay, 2003;Lemaitre and Poupin, 2003;Poupin and Malay, 2009;Negri et al., 2014), and has been considered relevant in the identification of other decapod species (see Poore et al., 2017 for review).In addition, different color patterns may have significance in the reproductive context, such as being decisive in mate choice (e.g., Detto et al., 2006), which may therefore be a barrier of reproductive isolation (Malay and Paulay, 2009).Furthermore, camouflage by coloration is considered a usual anti-predator strategy and might be related to behavioral and morphological mechanisms (e.g.Hamasaki et al., 2017).
The hermit crab genus Calcinus Dana, 1851 includes 43 species (McLaughlin et al., 2010) and is characterized by having morphologically similar species, causing misinterpretations and misidentifications (Haig and McLaughlin, 1983).Even though changes in color are known for some Calcinus species, colors might change markedly and fast during preservation (Haig and McLaughlin, 1983) and this is the main reason why color is frequently rejected in some systematic studies of decapod crustaceans (e.g.Chassard-Bouchaud, 1965;Malay and Paulay, 2009).
In this context, as color patterns have been considered important taxonomic characters for Calcinus species, we sought to evaluate if color differences between some specimens of Calcinus tibicen (Herbst, 1761) co-occurring in the same locality were based on high genetic differences.
General molecular analysis protocols followed Schubart et al. (2000), with modifications according to Mantelatto et al. (2006).For DNA extraction, salting-out extraction method (Miller et al., 1988) was used, with changes aiming suitability to the material.The primers COH6 (5'-TADACTTCDGGRTGDCCAAARAAYCA-3) and COL6b (5'-ACAAATCATAAAGATATYGG-3') (Schubart and Huber, 2006) were used in amplification by means of a Polymerase Chain Reaction (PCR) (Sambrook et al., 1989).Thermocycling conditions included: initial denaturation (2 min at 94°C), 35 annealing cycles (94°C for 30 s; 50°C for 30 s; 72°C for 1 min); final extension (2 min at 72°C).PCR products were purified using the kit SureClean and sequenced with the ABI Big Dye Terminator Mix (Applied Biosystem, Carlsba, CA, USA) in an ABI 3730xl DNA Analyzer (Applied Biosystems automated sequencer).Sequences were edited, and a consensus sequence was obtained from both strands (forward and reverse directions), using the program BioEdit 7.2.5.0 (Hall, 1999).Sequences (608 base pairs) were aligned via Clustal W (Thompson et al., 1994) with interface to BioEdit 7.2.5.0 (Hall, 1999).Matrixes of genetic divergence were computed in the program Molecular Evolutionary Genetics Analysis (MEGA) 6, using Kimura 2-parameters (Kimura, 1980).A Neighbor Joining tree was constructed using the software MEGA 6 (Tamura et al., 2013) based on genetic distance.In this analysis we used the following species as outgroups: Calcinus californiensis Bouvier, 1898 (GenBank access: KT89759), Calcinus explorator Boone, 1930 (GenBank Our results showed high levels of genetic homogeneity (0.2-1.1% of variation), and the Neighbor Joining tree based on the genetic distance also pointed out no isolation amongst the differently colored specimens -geographically separated by more than 2.500 km -of C. tibicen from Pernambuco and São Paulo (Fig. 1C); the opposite, i.e. high levels of variation (15.5-23.3%),was observed when comparing these colored individuals with the outgroups.These Low genetic variation in colored specimens of C. tibicen Nauplius, 26: e2018008 data indicate that these patterns are polymorphisms of the species as reported for other decapods (e.g.Terossi and Mantelatto, 2010), and are probably related to ecological factors, but hardly to a reproductive barrier.
Here we suggest some ecological factors, nonexclusive, probably related to the color patterns of the chelae: thermoregulation, intraspecific communication, and camouflage (Endler, 1978).The tons of the animals, in general, tend to be similar to their backgrounds (e.g.Poulton, 1890;Duarte et al., 2016), and in this case, some of the animals were collected in association and/or close to the red or green algae banks (FLM, pers.obs.).It means that individuals with orange or green chelae inhabit the same region, nevertheless, they can occupy different habitats with distinct background coloration, what might represent camouflage aspects, as already reported for other decapods (e.g.Bauer, 1981;Duarte et al., 2016).Morphology, coloration, and behavior can be linked to different ecological and evolutionary predation pressure under the same habitat (Hacker and Madin, 1991), related to predator color vision and predator color acuity (Endler, 1978).Natural predators of C. tibicen in this situation might be the fish Diodon hystrix Linnaeus, 1758 (Randall, 1967), and invertebrates such as Octopus vulgaris Cuvier, 1797 and Octopus briareus Robson, 1929(Gilchrist, 2003).Then, the variation in the chelae continued in C. tibicen's populations representing adaptive significance against different predators and the color patterns can be an interesting way of camouflage against visually-hunting predators (Bauer, 1981;Hamasaki et al., 2017).Due to the relationship with the substrate, it can also be conjectured that the tons of colors are also related to the kind of nutrition of the specimens.
Therefore, although in genus Calcinus color patterns are taken to species-specific recognition or identification, in the case of C. tibicen evaluated here, this characteristic may not be used (in opposition to what was reported in Salmon et al., 1979;Bruce, 1978;Malay and Paulay, 2009;Negri et al., 2014;Hamasaki et al., 2017).Both colors of chelae occur within the same species, which was represented by the low rates of genetic variation among them and when compared with the genetic range of variation (0-9.9%)observed along the entire C. tibicen distribution in the Western Atlantic (Mandai et al., unpublished data).Some causes related to these differences in colors of the chelae, orange or green, were inferred here; however, more studies about C. tibicen's ecology and feeding might be taken to better understand this intriguing pattern in some regions of its distribution.