Unraveling the true identity of the pugilator (Crustacea: : ) and study of the associated complex

Bruno Alm´on*1, Jacinto P´erez2, Eva Garc´ıa-Isarch3, J. Enrique Garc´ıa-Raso4 and Jose A. Cuesta5

*Corresponding author: [email protected] 1Instituto Espa˜nolde Oceanograf´ıa,Centro Oceanogr´aficode Vigo. Subida a Radio Faro, 50-52 36390 Vigo, Spain. 2Grupo de Estudo do Medio Mari˜no(GEMM), Puerto Deportivo s/n. 15960 Ribeira. A Coru˜na.Spain 3Instituto Espa˜nolde Oceanograf´ıa,C.O. de C´adiz.Muelle de Levante s/n, 11006, C´adiz,Spain 4Universidad de M´alaga, Fac. de Ciencias, Dep. Biolog´ıaAnimal, Campus Teatinos, s/n, 29071, M´alaga,Spain. 5Instituto de Ciencias Marinas de Andaluc´ıa,ICMAN-CSIC, Avenida Rep´ublicaSaharaui, 2, 11519 Puerto Real, C´adiz,Spain

Keywords: Diogenes, , Iberian Peninsula, DNA, Paguroidea

Abstract

Diogenes pugilator (Roux, 1829) is a widely distributed species which has been recorded profusely since its description based on specimens from the French Mediterranean. Its actual theoretical distribution covers all east-Atlantic shores, the Mediterranean and the Black Seas. It is very common in shallow waters from 0 to 40 m, especially in soft bottoms and sand beaches, where it can be easily spot, with a extremely long left chelae being dragged on the substrate. During the past decades, several morphotypes have been described based on notorious morphologic variations of different body parts, leading in some cases to the description of new species that years later have been largely synonymized to the original D. pugilator from Roux(1829). In his work, Roux gives a short description of the species along with a plate that, although it describes well the general form of the body of the species, it is not detailed enough to be able to compare certain important characteristics. Unfortunately, the type specimens of the Roux’s species seem to have disappeared after his death, so the clarification of the true identity of D. pugilator remains unclear.

Materials and Methods

In the framework of a more global study devoted to the objective of reviewing Iberian species of hermit crabs both from the morphological and molecular point of view, a review of several specimens identified as D. pugi- lator has been conducted, leading to the identification of a certain number of well-defined morphotypes, all previously included in the nominal species. Specimens studied have been collected by a variety of methods and come from different sources, including Biological Reference Colections (Colecci´onde Crust´aceosDec´apodos y Estomat´opodos del Centro Oceanogr´aficode C´adizdel IEO- CCDE-IEOCD, “Colecci´onBiol´ogicade Referen- cia del Institut de Ci`enciesdel Mar”- ICM-CSIC and the personal collections from Jos´eEnrique Garc´ıa-Raso from M´alagaUniversity, Jose A. Cuesta from ICMAN-CSIC, and Christoph Schubart from the University of Regensburg, Germany), from scuba diving, and from scientific cruises devoted to the evaluation of fisheries stocks (ARSA 2017-2018 and MEDITS 2017-2018). Samples were studied under the stereomicroscope and then photographed and sketched to define the main traits defining each of the morphotypes. Drawings were

1 then digitalized in Inkskape, with the aid of a digital tablet. Molecular samples were then taken from at least a pair of individuals from each of the species, one male and one female. Sequences of two mitochondrial genes (16S and COI) were obtained as well as others from GenBank that were also included in the comparative. A complete Maximum Likelihood phylogenetic tree was obtained for the available species of the Diogenes along with other species of the family Diogenidae in order to contextualize the phylogenetic position of the species of Diogenes and validate morphological differentiation.

Results and Discussion

Preliminary phylogenetic analysis agrees with the morphological delimitation of the morphotypes, suggesting that D. pugilator is in fact a species complex composed of several well-delimited species, and that all previous records of D. pugilator should be revised under the light of the new information. The discussion about which of the identified morphotypes correspond to the original from Roux(1829), is still unresolved due to the absence of type specimens. Nevertheless, the revision of the synonymized species preserved in the Museum Nationale d’Histoire Naturelle in Paris, along with an increasing sampling effort oriented to the area where the type specimens where initially caught, aims to serve as a clue to elucidate the true identity of the Roux species. Unraveling the true identity of D. pugilator is one of the most urgent tasks within this project, but it also opens a series of new challenges, since as the result of the designation of neotypes, other morphotypes need to be addressed and described in detail, updating the biological and distributional information associated with each of them. Once this task is finished, a complete revision of previous records and synonymies for this species should be also conducted and old names, perhaps, resurrected. Nevertheless, although this is still a work in progress, preliminary results suggest that specimens from Atlantic coast of the Iberian peninsula are not assignable to Roux species, which is more likely to be restricted to the Mediterranean Sea. Notwithstanding, two different morphotypes are also present in Iberian Atlantic waters being one of them associated with northern latitudes and the second with southern distribution. There are still many questions to solve around this problem, but it is important to reach an consensus about the definition of the species, given that the absence of original material, can lead to the perpetuation of the misidentifications making studies where these species are involved unreliable and the evaluation of the Diogenes spp. populations status, consistently overestimated. See Figure1 and Figure2.

2 ER146 ER142 Diogenes pugilator ER144 Diogenes pugilator 8 ER140 Diogenes pugilator KT209045.1 Diogenes pugilator 7 KT208608.1 Diogenes pugilator

24 KT208866.1 Diogenes pugilator KT208586.1 Diogenes pugilator 24 KT209297.1 Diogenes pugilator KT208810.1 Diogenes pugilator 24 ERMI11 COI Diogenes pugilator ERMI10 COI Diogenes pugilator 38 ERMI04 COI Diogenes pugilator 33 ERMI06 COI Diogenes pugilator 52 ER126+COH6 Diogenes pugilator 100 ERMI07 COI Diogenes pugilator ER123+COH6 Diogenes pugilator 48 ER128+COH6 Diogenes sp

23 ERMI09 COI Diogenes sp 18 ERMI02 COI Diogenes sp 100 ER127b+COH6 Diogenes sp

33 ERMI08 COI Diogenes sp 24 ERMI01 COI Diogenes sp 8 25 ER136 Diogenes sp

28 ERMI03 COI Diogenes sp

88 ER125+COH6 Diogenes sp 46 ER124 Diogenes sp

100 ERMI13 COI Cestopagurus timidus ERMI05 COI Cestopagurus timidus

99 KJ150701.1 Diogenes canaliculatus 100 10 KM043476.1 Diogenes canaliculatus KM043474.1 Diogenes merguiensis

100 ER135 Diogenes sp1 45 ER66 Diogenes sp1

0 100 ER137 Diogenes sp2 ER68 Diogenes sp2 97 ER139 Diogenes sp2 92 ER62a Diogenes sp2 35 16 ER70a Diogenes sp2 27 ER64 Diogenes sp2

100 KM043472.1 Diogenes alias

77 KJ150705.1 Diogenes violaceus KM043473.1 Diogenes dubius 94 100 KJ150703.1 Diogenes miles 1 KM043475.1 Diogenes manaarensis 100 KJ150704.1 Diogenes planimanus 4 100 ER11+COH6 Petrochirus pustulatus ER03+COH6 Petrochirus pustulatus ER106+COH6 Dardanus pectinatus 2 54 51 ER105+COH6 Dardanus arrosor 90 ER43+COH6 Dardanus arrosor 100 ER103+COH6 Dardanus pectinatus 1 94 ER101+COH6 Dardanus arrosor ER20+COH2198 Diogenes ovatus KJ150702.1 Diogenes custos 17

100 100 61

100 100 20

58 100

100 KF808160.1 Diogenes pugilator

0.020

Figure 1: Part of the resulting COI phylogenetic tree showing the different resulting groups involving Diogenes species.

3 Figure 2: Original illustration from Roux(1829), describing the general morphology of the species.

Acknowledgments

We want to thank Christoph Schubart, Carlos Farias and Pere Abell´o,the cession of specimens from their samplings and scientific surveys.

References

Garc´ıa,E., Mu˜noz,I., 2013. Collection of decapod and stomatopod from oceanographic center of cadiz. URL: www.gbif.es. Olivas, F.J., 2007. Colecci´onbiol´ogicade referencia del institut de ci`encies del mar- icm-csic. URL: http: //cbr.icm.csic.es/ca/site-page/cerca. Roux, P., 1829. Crustac´esde la Mediterran´eeet de son littoral. Annales des Sciences Naturelles.

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