Abstract

Apart from traditional characters, other data have been used for taxonomy, like color patterns. Based on the different 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 flow between specimens with different color patterns, which are polymorphisms and might be related to the kind of nutrition as well different ecological and evolutionary predation characteristics.

Key words:
Anomura; COI gene; coloration; Diogenidae; polymorphism

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, 1986Knowlton, N. 1986. Cryptic and sibling species among the Decapod Crustacea. Journal of Crustacean Biology, 6: 356-363.; Malay and Paulay, 2009Malay, M.C.M.D. and Paulay, G. 2009. Peripatric speciation drives diversification and distributional pattern of reef hermit crabs (Decapoda: Diogenidae: Calcinus). The Society for the Study of Evolution, 64: 634-662.). 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., 1979Salmon, S.D.; Ferris, D.; Johnston, G.; Hyatt, G. and Whitt, G.S. 1979. Behavioral and biochemical evidence for species distinctiveness in the fiddler crabs, Uca speciosa and U. spinicarpa. Evolution, 33: 182-191.; Williams and Felder, 1986Williams, A.B. and Felder, D.L. 1986. Analysis of stone crabs: Menippe mercenaria (Say), restricted, and a previously unrecognized species described (Decapoda: Xanthidae). Proceedings of the Biological Society of Washington, 99: 517-543. ; Bruce, 1978Bruce, A.J. 1978. The evolution and zoogeography of shallow-water tropical shrimps. Information Series, Department of Scientific and Industrial Research, New Zealand, 137: 337-355.; Malay and Paulay, 2009Poupin, J. and Malay, M.C.M.D. 2009. Identification of a Ciliopagurus strigatus (Herbst, 1804) species-complex, with description of a new Ciliopagurus from French Polynesia (Decapoda, Anomura, Diogenidae). Zoosystema , 31: 209-232.; Negri et al., 2014Negri, M.; Lemaitre, R. and Mantelatto, F.L. 2014. Molecular and morphological resurrection of Clibanarius symmetricus (Randall, 1840), a cryptic species hiding under the name for the “thinstripe” hermit crab C. vittatus (Bosc, 1802) (Decapoda: Anomura: Diogenidae). Journal of Crustacean Biology , 34: 848-861.; Hamasaki et al., 2017Hamasaki, K.; Tsuru, T.; Sanda, T.; Fujikawa, S.; Dan, S. and Kitada, S. 2017. Ontogenetic change of body color patterns in laboratory-raised juveniles of six terrestrial hermit crab species. Zootaxa, 4226: 521-545.).

Among hermit crabs, color pattern differentiation is quite significant between cryptic species (e.g. Asakura and Paulay, 2003Asakura, A. and Paulay, G. 2003. Pylopaguropsis lemaitrei, a new species of hermit crab (Decapoda: Anomura: Paguridae) from French Polynesia. Crustacean Research, 32: 13-25.; Lemaitre and Poupin, 2003Lemaitre, R. and Poupin, J. 2003. A strikingly coloured new species of Paragiopagurus Lemaitre, 1996 (Crustacea: Decapoda: Anomura: Parapaguridae) from French Polynesia. Zootaxa , 386: 1-11.; Poupin and Malay, 2009Poupin, J. and Malay, M.C.M.D. 2009. Identification of a Ciliopagurus strigatus (Herbst, 1804) species-complex, with description of a new Ciliopagurus from French Polynesia (Decapoda, Anomura, Diogenidae). Zoosystema , 31: 209-232.; Negri et al., 2014Negri, M.; Lemaitre, R. and Mantelatto, F.L. 2014. Molecular and morphological resurrection of Clibanarius symmetricus (Randall, 1840), a cryptic species hiding under the name for the “thinstripe” hermit crab C. vittatus (Bosc, 1802) (Decapoda: Anomura: Diogenidae). Journal of Crustacean Biology , 34: 848-861.), and has been considered relevant in the identification of other decapod species (see Poore et al., 2017Poore, A.G.B.; Ahyong, S.T.; Lowry, J.K. and Sotk, E.E. 2017. Plant feeding promotes diversification in the Crustacea. PNAS, 114: 8829-8834. 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., 2006Detto, T.; Backwell, P.R.; Hemmi, J.M. and Zeil, J. 2006. Visually mediated species and neighbor recognition in fiddler crabs (Uca mjoebergi and Uca capricornis). Proceedings of the Royal Society B: Biological Sciences, 273: 1661-1666.), which may therefore be a barrier of reproductive isolation (Malay and Paulay, 2009Poupin, J. and Malay, M.C.M.D. 2009. Identification of a Ciliopagurus strigatus (Herbst, 1804) species-complex, with description of a new Ciliopagurus from French Polynesia (Decapoda, Anomura, Diogenidae). Zoosystema , 31: 209-232.). 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., 2017Hamasaki, K.; Tsuru, T.; Sanda, T.; Fujikawa, S.; Dan, S. and Kitada, S. 2017. Ontogenetic change of body color patterns in laboratory-raised juveniles of six terrestrial hermit crab species. Zootaxa, 4226: 521-545.).

The hermit crab genus Calcinus Dana, 1851 includes 43 species (McLaughlin et al., 2010McLaughlin, P.A.; Komai, T.; Lemaitre, R. and Rahayu, D.L. 2010. Annotated checklist of Anomuran Decapod crustaceans of the world (exclusive of the Kiwaoidea and families Chirostylidae and Galatheidae of the Galatheoidea). Part I - Lithodoidea, Lomisoidea and Paguroidea. The Raffles Bulletin of Zoology, Supplement 23: 5-107.) and is characterized by having morphologically similar species, causing misinterpretations and misidentifications (Haig and McLaughlin, 1983Haig, J. and Mclaughlin, P.A. 1983. New Calcinus species (Decapoda: Anomura: Diogenidae) from Hawaii, with a key to the local species. Micronesica, 19: 107-121.). Even though changes in color are known for some Calcinus species, colors might change markedly and fast during preservation (Haig and McLaughlin, 1983Haig, J. and Mclaughlin, P.A. 1983. New Calcinus species (Decapoda: Anomura: Diogenidae) from Hawaii, with a key to the local species. Micronesica, 19: 107-121.) and this is the main reason why color is frequently rejected in some systematic studies of decapod crustaceans (e.g. Chassard-Bouchaud, 1965Chassard-Bouchaud, C. 1965. L’adaptation chromatique chez les Natantia (Crustacés Décapodes). Paris, Robin & Mareuge, 104p.; Malay and Paulay, 2009Malay, M.C.M.D. and Paulay, G. 2009. Peripatric speciation drives diversification and distributional pattern of reef hermit crabs (Decapoda: Diogenidae: Calcinus). The Society for the Study of Evolution, 64: 634-662.).

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.

We evaluated the genetic distance for the cytochrome oxidase subunit I (COI) mitochondrial gene between six differently colored individuals (orange and green) from the states of Pernambuco (PE, 08°16’15.56”S 34°56’40.34”W) and São Paulo (SP, 23°25’20.03”S 45°3’42.61”W), Brazil (Fig. 1A, B) [PE orange: CCDB 5327 - GenBank accesses KT897571, KT897572; PE green: CCDB 5328 - GenBank accesses KT897552, KT897570; SP orange: CCDB 0769 - GenBank access KT897493; SP green: CCDB 0778 - GenBank access KT897522]. The identification of the specimens was confirmed based on morphological characters (Melo, 1999Melo, G.A.S. 1999. Manual de identificação dos Crustacea Decapoda do litoral brasileiro: Anomura, Thalassinidea, Palinuridea, Astacidea. São Paulo, Plêidae, 551p.). Here we report that the green pattern has changed its color after preservation in 70% ethanol.

Figure 1
Specimens of Calcinus tibicen (Herbst, 1761) from two Brazilian localities (PE: Pernambuco and SP: São Paulo), showing differences in the color patterns of the chelae in life. (A) green claw male, CCDB 5328, scale bar = 10 mm. (B): orange claw male, CCDB 5327, scale bar = 11 mm (both from Pernambuco). (C) Neighbor Joining tree of C. tibicen specimens based on genetic distance for COI gene. Photos by R.C. Buranelli.

General molecular analysis protocols followed Schubart et al. (2000Schubart, C.D.; Neigel, J.E. and Felder, D.L. 2000. Use of the mitochondrial 16S rRNA gene for phylogenetic and population studies of Crustacea. In: J.C. von Vaupel Klein and F.R. Schram (eds), The Biodiversity Crisis and Crustacea - Proceedings of the 4th International Crustacean Congress, Balkema, Rotterdam, Brookfield, The Netherlands. Crustacean Issues, 12: 817-830. ), with modifications according to Mantelatto et al. (2006Mantelatto, F.L.; Robles, R.; Biagi, R. and Felder, D.L. 2006. Molecular analysis of the taxonomic and distributional status based for the hermit crab genera Loxopagurus Forest, 1964, and Isocheles Stimpson, 1858 (Decapoda, Anomura, Diogenidae). Zoosystema, 28: 495-506.). For DNA extraction, salting-out extraction method (Miller et al., 1988Miller, S.A.; Dykes, D.D. and Polesky, H.F. 1988. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Research, 16: 1215. ) was used, with changes aiming suitability to the material. The primers COH6 (5’-TADACTTCDGGRTGDCCAAARAAYCA-3) and COL6b (5’- ACAAATCATAAAGATATYGG-3’) (Schubart and Huber, 2006Schubart, C.D. and Huber, M.G.J. 2006. Genetic comparisons of german populations of the stone crayfish, Austropotamobius torrentium (Crustacea: Astacidae). Bulletin Francais de la Peche et de la Pisciculture, 380: 1019-1028. ) were used in amplification by means of a Polymerase Chain Reaction (PCR) (Sambrook et al., 1989Sambrook, J.; Fritsch, E.F. and Maniatis, T. 1989. In vitro amplification of DNA by the Polymerase Chain Reaction, p. 2-35. In: J. Sambrook; E.F. Fritsch and T. Maniatis (eds), Molecular cloning: a laboratory manual. New York, Cold Spring Harbor Laboratory Press.). 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, 1999Hall, T.A. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series, 41: 95-98. ). Sequences (608 base pairs) were aligned via Clustal W (Thompson et al., 1994Thompson, J.D.; Higging, D.G. and Gibson, T.J. 1994. CLUSTALW: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting specific gap penalties and weight matrix choice. Nucleic Acids Research , 22: 4673-4680.) with interface to BioEdit 7.2.5.0 (Hall, 1999Hall, T.A. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series, 41: 95-98. ). Matrixes of genetic divergence were computed in the program Molecular Evolutionary Genetics Analysis (MEGA) 6, using Kimura 2-parameters (Kimura, 1980Kimura, N. 1980. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. Journal of Molecular Evolution, 16: 111-120.). A Neighbor Joining tree was constructed using the software MEGA 6 (Tamura et al., 2013Tamura, K.; Stecher, G.; Peterson, D.; Filipski, A. and Kumar, S. 2013. MEGA6: Molecular Evolution ary Genetics Analysis Version 6.0. Molecular Biology and Evolution , 30: 2725-2729. ) 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 access: FJ620320), Calcinus laevimanus (Randall, 1840) (GenBank access: FJ620270), Calcinus obscurus Stimpson, 1859 (GenBank access: KT897589) and Calcinus tubularis (Linnaeus, 1767) (GenBank access: KT897590).

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 data indicate that these patterns are polymorphisms of the species as reported for other decapods (e.g. Terossi and Mantelatto, 2010Terossi, M. and Mantelatto, F.L. 2010. Sexual ratio, reproductive period and seasonal variation of the gonochoric shrimp Hippolyte obliquimanus (Caridea: Hippolytidae). Marine Biology Research , 6: 213-219.), and are probably related to ecological factors, but hardly to a reproductive barrier.

Here we suggest some ecological factors, non-exclusive, probably related to the color patterns of the chelae: thermoregulation, intraspecific communication, and camouflage (Endler, 1978Endler, J.A. 1978. A Predator’s View of Animal Color Patterns. p. 319-364. In: M.K. Hecht; W.C. Steere and B. Wallace (eds), Evolutionary Biology, Vol. 11. Boston, Springer.). The tons of the animals, in general, tend to be similar to their backgrounds (e.g. Poulton, 1890Poulton, E.B. 1890. The colors of animals, their meaning and use, especially considered in the case of insects. New York, D. Appleton and Company, 365p.; Duarte et al., 2016Duarte, R.C.; Stevens, M. and Flores, A.A.V. 2016. Shape, colour plasticity, and habitat use indicate morph-specific camouflage strategies in a marine shrimp. BMC Evolutionary Biology, 16: 218-232.), 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, 1981Bauer, R.T. 1981. Color Patterns of the Shrimps Heptacarpus pictus and H. paludicola (Caridea: Hippolytidae). Marine Biology, 64: 141-152.; Duarte et al., 2016Duarte, R.C.; Stevens, M. and Flores, A.A.V. 2016. Shape, colour plasticity, and habitat use indicate morph-specific camouflage strategies in a marine shrimp. BMC Evolutionary Biology, 16: 218-232.). Morphology, coloration, and behavior can be linked to different ecological and evolutionary predation pressure under the same habitat (Hacker and Madin, 1991Hacker, S.D. and Madin, L.P. 1991. Why habitat architecture and color are important to shrimps living in pelagic Sargassum: use of camouflage and plant-part mimicry. Marine Ecology Progress Series, 70: 143-155.), related to predator color vision and predator color acuity (Endler, 1978Endler, J.A. 1978. A Predator’s View of Animal Color Patterns. p. 319-364. In: M.K. Hecht; W.C. Steere and B. Wallace (eds), Evolutionary Biology, Vol. 11. Boston, Springer.). Natural predators of C. tibicen in this situation might be the fish Diodon hystrix Linnaeus, 1758 (Randall, 1967Randall, J.E. 1967. Food habits of reef fishes of the West Indies. Studies in Tropical Oceanography, 5: 665-847.), and invertebrates such as Octopus vulgaris Cuvier, 1797 and Octopus briareus Robson, 1929 (Gilchrist, 2003Gilchrist, S.L. 2003. Hermit crab population ecology on a shallow coral reef (Bailey’s Cay, Roatan, Honduras): octopus predation and hermit crab shell use. Memoirs of Museum Victoria, 60: 35-44.). 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, 1981Bauer, R.T. 1981. Color Patterns of the Shrimps Heptacarpus pictus and H. paludicola (Caridea: Hippolytidae). Marine Biology, 64: 141-152.; Hamasaki et al., 2017Hamasaki, K.; Tsuru, T.; Sanda, T.; Fujikawa, S.; Dan, S. and Kitada, S. 2017. Ontogenetic change of body color patterns in laboratory-raised juveniles of six terrestrial hermit crab species. Zootaxa, 4226: 521-545.). Due to the relationship with the substrate, 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., 1979Salmon, S.D.; Ferris, D.; Johnston, G.; Hyatt, G. and Whitt, G.S. 1979. Behavioral and biochemical evidence for species distinctiveness in the fiddler crabs, Uca speciosa and U. spinicarpa. Evolution, 33: 182-191.; Bruce, 1978Bruce, A.J. 1978. The evolution and zoogeography of shallow-water tropical shrimps. Information Series, Department of Scientific and Industrial Research, New Zealand, 137: 337-355.; Malay and Paulay, 2009Malay, M.C.M.D. and Paulay, G. 2009. Peripatric speciation drives diversification and distributional pattern of reef hermit crabs (Decapoda: Diogenidae: Calcinus). The Society for the Study of Evolution, 64: 634-662.; Negri et al., 2014Negri, M.; Lemaitre, R. and Mantelatto, F.L. 2014. Molecular and morphological resurrection of Clibanarius symmetricus (Randall, 1840), a cryptic species hiding under the name for the “thinstripe” hermit crab C. vittatus (Bosc, 1802) (Decapoda: Anomura: Diogenidae). Journal of Crustacean Biology , 34: 848-861.; Hamasaki et al., 2017Hamasaki, K.; Tsuru, T.; Sanda, T.; Fujikawa, S.; Dan, S. and Kitada, S. 2017. Ontogenetic change of body color patterns in laboratory-raised juveniles of six terrestrial hermit crab species. Zootaxa, 4226: 521-545.). 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.

Acknowledgements

This article was part of the Bachelor’s thesis of SSM supported by scientific fellowship of Fundação de Amparo à Pesquisa do Estado de São Paulo - FAPESP (2014/10639-1). Major financial support was provided by FAPESP (2002/08178-9, Temático Biota 2010/50188-8; Coleções Científicas 2009/54931-0; APQ 2016/50376-5), Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq (Procs. 473050/2007-2, 471011/2011-8, 504322/2012-5; Research Scholarship PQ 302748/2010-5, 304968/2014-5) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES (Ciências do Mar II 2005/2014 - 23038.004308/2014-14) to FLM. RCB thanks the Post-Doctoral fellowship by CNPQ (PROTAX 150462/2016-6). We thank anonymous reviewers for their suggestions that help to improve the quality of this paper during revision process. The authors, especially FLM, are honored to recognize the many achievements of our colleague, Ludwig Buckup, to development of carcinology in Brazil and for this reason dedicate this contribution for this commemorative edition to him.

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