- Open Access
DNA barcoding of the spider crab Menaethius monoceros (Latreille, 1825) from the Red Sea, Egypt
Journal of Genetic Engineering and Biotechnology volume 19, Article number: 42 (2021)
Most spider crab species inhabiting the Red Sea have not been characterized genetically, in addition to the variation and complexity of morphological identification of some cryptic species. The present study was conducted to verify the identification of two morphotypes of the spider crab Menaethius monoceros (Latreille, 1825) in the family Epialtidae Macleay, 1838, collected from the Red Sea, Egypt. DNA barcoding of two mitochondrial markers, cytochrome oxidase subunit I (COI) and 16S, was used successfully to differentiate between these morphotypes.
DNA barcoding and genetic analyses combined with morphological identification showed that the two morphotypes were clustered together with low genetic distances ranged from 1.1 to 1.7% COI and from 0.0 to 0.06% 16S. Hence, this morphological variation is considered as individual variation within the same species.
The present study successively revealed that genetic analyses are important to confirm the spider crab’s identification in case of morphological overlapping and accelerate the accurate identification of small-sized crab species. Also, DNA barcoding for spider crabs is important for better future evaluation and status records along the Red Sea coast.
The horny spider crab genus Menathius H. Milne Edwards, 1834 (Epialtidae Macleay, 1838) contains only two species: Menaethius monoceros (Latreille,1825) and Menaethius oriantalis (Sakai, 1969) [1,2,3]. They are common in the Indo-West Pacific extending from the Red Sea, Eastern Africa, and the Indian Ocean to Japan in the Pacific Ocean . Recently, M. monoceros had its distribution extended and was recorded from the Mediterranean Sea by . These species are known to inhabit weedy intertidal rocks in shallow waters [1, 2, 5, 6]. This genus can be distinguished by the following morphological characters: the presence of two small lobes or tubercles on the branchial margins, a small post-orbital lobe, propodi of the first walking leg is smooth ventrally, and with sexes similar in form. It is easy to differentiate M. monoceros from M. oriantalis by the presence of a slender rostrum, basally narrow; the dorsal branchial region has tubercles or rounded elevation; and walking legs are not carinate in M. monoceros. On the other hand, carinate walking legs are present in M. orientalis . However, this genus has a geographical variation, as many authors have described several species which synonymized under M. monoceros  and listed by  treated in the list of Griffin and Tranter as synonyms .
Majidae is considered as a diverse group within brachyuran crabs, containing over 800 species, and was recently transferred to the superfamily Majoidea Samouelle, 1819 [3, 8,9,10]. Despite the revision of the majoid crabs by many authors [1,2,3, 11,12,13,14] throughout the Indo-West Pacific region and the list of  from the Arabian Gulf, some genera and species still have taxonomic confusion, and many workers have difficulty in identification using the keys within subfamilies to recognize the genera and species.
The eastern Egyptian coast of the Red Sea includes the eastern and western coasts of the Suez Gulf and the western Aqaba Gulf coast, with a total length of about 1300 km . The Suez Gulf is considered as the boundary between Asia and Africa having an important role in the faunal migration between the Red Sea and the Mediterranean Sea  via the Suez Canal. The coasts of the Red Sea are represented by several habitats: soft, rocky, and coral reef habitats. These different habitats comprise suitable substrates as refuges and preferable habitats for different marine invertebrates.
The crabs of the Red Sea were listed and reviewed by [18,19,20,21,22,23,24,25]. Other later workers reviewed the majoids  and listed 32 species, as well as accounts of Red Sea majoids by [1, 26] who listed 46 spider crab species within Majoidea including 12 species in the family Epialtidae: Acanthonyx elongatus Miers, 1877; A. dentatus H. Milne Edwards, 1834; Huenia heraldica (De Haan, 1837) [= Maja (Huenia) proteus De Haan, 1839]; Menaethiops contiguicornis (Klunzinger, 1906); M. dubius Balss, 1929; M. ninii Guinot, 1962; M. nodulosus (Nobili, 1905); Menaethius monoceros (Latreille, 1825); Perinia tumida Dana, 1851; Simocarcinus pyramidatus (Heller, 1861); S. simplex (Dana, 1852); and Xenocarcinus tuberculatus White, 1847.
The recent revisions of brachyuran crabs depending only on morphological descriptions have many issues [27, 28]. And therefore, the use of DNA barcoding can help accelerate the identification of confused and even cryptic species [27, 29]. Molecular phylogenetics is a valuable tool to study the morphological evolution of decapod crustaceans, which may reflect their behaviors and distributions [27, 30,31,32,33].
Furthermore, using mitochondrial cytochrome oxidase subunit I gene in molecular studies in crustacean’s taxonomy is useful for species delimitation [29, 34]. Besides COI, the 16S rRNA gene is commonly used in constructing animal phylogeny because of the combination of the variable and conserved region with the same gene . Also, the sequence lengths obtained with these two markers are in the range of sequences available in the GenBank database. With this in mind, the present study applied the DNA barcoding information of the spider crab M. monoceros exclusively from the Red Sea, Egypt, and successfully used phylogenetic analyses to confirm two possible morphotypes.
A total of 20 specimens examined in the present study were collected from several locations across Red Sea Egyptian coasts: Hurghada (Marine Biology Station), Marsa Gabal El-Rosass, South Hammata, 9km north Marsa Alam, and south Bernis. Specimens were obtained attached to small weedy rocks in the shallow intertidal region.
DNA was extracted from pieces of walking leg tissue for each crab specimen using a Qiagen DNA extraction kit (Germany); the final observed DNA concentrations with Nanodrop measuring machine were 19.2–27.7 ng μl. PCR amplification of 16S and COI markers was performed with 20-μl PCR cocktail volumes using 40 cycles (20μl) of 94°C for 40 s (denaturation), 48°C for 70 s (annealing), and 72°C for 90 s (extension), run on a PCR thermal cycler. PCR products were observed on 1.5 % agarose gels. Subsequently, purification of the formed single-band PCR products was done using a mixture of shrimp alkaline phosphate (SAP) and exonuclease (EXO1) protocol (37° C for 20 min followed by 83°C for 30 min.). Sequences were obtained by sending the cleaned PCR products to FASMAC (Yokohama, Japan). The acquired sequences were assembled using Bioedit and aligned and analyzed using the Mega 7 program . The evolutionary history was inferred using the neighbor-joining method . The evolutionary distances were computed for CO1 sequences using the Tamura 3-parameter method  while for 16S sequences using the Kimura 2-parameter method . All accession numbers of the sequences used in genetic analyses are listed in Table 1. COI and 16S genes were amplified using universal primer sets following [40, 41], respectively.
Epialtidae Macleay, 1838
Menaethius H. Milne Edwards, 1834
Menaethius monoceros (Latreille, 1825)
Pisa monoceros Latreille, 1825:139-140.
Menaethius monoceros. Alcock, 1895: p. 197. -Klunzinger, 1906: p. 20. -Balss, 1924: p. 27. -Urita, 1926: p. 32. -Sakai, 1934: p. 294; 1936: p. 91, pl. 21, fig. 3; 1938: p. 263, pl. XXVI fig. 3 -Forest and Guinot, 1961:14, fig. 9a & b. -Sakai, 1965: 74-75, pi. 33 fig. 4. -Griffin, 1974:21.
RCAZUE.Crus-Br.26151.13, four females, Hurghada, Marine Biology Station, Red Sea, Egypt, 27° 17′ 18.9″ N, 33° 45′ 46.6″ E, and other additional specimens previously collected during the period from April 1996 to February 1997 from the Red Sea Egyptian coasts are listed in Table 2.
Despite the similarity between sexes in some characters for the genus Menaethius described by  and the agreement for some characters described by  especially in the rostrum (slender or some time bifurcated) which were similar to the present all specimens, there were two differences between the present specimens. We observed two morphotypes. The first morphotype consisted of two specimens (CL, 5.6 and 6.9) with slightly bifurcated rostrum in appearance and obtuse lobes or tubercles in the branchial margins versus the other morphotype (CL, 13.4 and 14.1) which had a rounded tip of the rostrum and obvious lobes or tubercles on the branchial margins. On the other hand, the comparison of all descriptions of synonymized names for M. monoceros includes a variety of morphological differences. Moreover, there is a variation in the morphology of this species across its geographical distribution , in addition to slight differences observed between two populations examined by  from two localities at the northern Red Sea. In addition, there has been a variation reported in carapace tuberculation in different sites (Figs. 1 and 2). All of these previous reasons make it difficult to accurately identify the morphotype of the present specimens, making DNA analyses important to reach a precise identification.
The genetic analyses of the two markers used, 16S and COI (Figs. 3 and 4 and Tables 3 and 4), revealed that the two closely related morphotypes of M. monoceros showed slight genetic differences. The COI neighbor-joining tree separated the two morphotypes, but the genetic differences were only 1.1 to 1.7% (Table 3). As shown in Table 4, the genetic distance for 16S sequences between the two morphotypes was negligible, ranging from 0.0 to 0.6%. These results indicate that the genetic differences are not enough to separate these morphotypes into different species based on a cutoff OTU threshold of 97% , and thus, I consider this variation as individual variation within the same species.
The present specimens were observed and obtained from weedy shallow intertidal small rocks at depths of 0–2m.
Larger specimens have a gray color on the legs and branchial regions with light orange color on the hepatic and cardiac prominence, but the smaller specimens are white to creamy all over the carapace and legs.
Red Sea, throughout the Indo-West Pacific from South Africa to Japan, Australia, and Tahiti, recently recorded from the Mediterranean Sea.
Two or more species imprecisely classified or distinguished morphologically under one species name usually called cryptic species . Moreover, the great diversity among the Indo-West Pacific fauna of majoid crabs was treated morphologically by [1, 2]. Despite the provided key by Grifin and Tranter, many problems still face many workers in the identification of majoid crabs, and as such, this confusion has caught the attention of many workers [12,13,14].
The genus Menaethius has a variation in characters described by  in between its only two species (M. monoceros and M. oriantalis). The dorsal surface of carapace is mounted with a variable number of tubercles on the gastric and cardiac regions, the male pseudorostrum is usually relatively longer than females, and the tip is somewhat rounded and sometimes bifurcated. The branchial margins often have two teeth and sometimes with numerous tubercles. Some variation of the previous characters was also observed in the present studied specimens (Figs. 1 and 2) which leads to doubtful morphological identification and needs confirmation with genetic analyses.
It is known that delimitation of single species for discovering new species is a modern topic of discussion in systematics . Thus, DNA barcoding for animal species is considered as a somewhat new and important taxonomic tool . Moreover, DNA barcoding can speed the morphological identification of described species . The present study investigated four specimens resembling the original description with variation in some characters and appearance of two morphotypes. Molecular analyses showed that the genetic distance difference between them ranged from 0.6 to 1.7% (Tables 3 and 4), which is low and not enough to separate them into different species. However, the genetic differences in the distance between the present studied specimen’s sequences from those from Shimoda, Japan (EU682804, EU682856), obtained from GenBank ranged between 1.4 and 2.1%, for 16S marker and between 1.42 and 4.8% in COI. These larger differences may be attributed to the geographical variation between both populations (Red Sea versus Pacific). Finally, DNA barcoding proved to increase the identification speed of small crab species, and thus, we consider it has a potential to be an important tool combined with morphology in taxonomic studies.
Most small-sized crab species inhabiting the Red Sea and associated with seaweeds are difficult to identify morphologically, and thus, the present study used two DNA markers to confirm the identity of two morphotypes of the spider crab Menaethius monoceros. DNA barcoding and phylogeny revealed that there was a variation within the same species. Also, the present study provides baseline DNA data for one species inhabiting the Red Sea, which has recently been reported as migrated to the Mediterranean Sea. Our data will be important in further future investigations.
Availability of data and materials
The datasets used and analyzed during the current study are presented in the article and additional genetic data (sequences) available and deposited in the GenBank with an accession number.
Cytochrome oxidase subunit I
Ribosomal subunit rRNA
Griffin DJG, Tranter HA (1974) Spider crabs of the family Majidae (Crustacea: Decapoda: Brachyura) from the Red Sea. Israel J Zool. 23:162–198
Griffin DJG, Tranter HA (1986) The Decapoda Brachyura of the Siboga expedition, part VIII. Majidae. Siboga-Expeditie Monograph. 39(C4). Livraision. 148:1–335
Ng PKL, Guinot D, Davie PJF (2008) Systema Brachyurorum: part I. An annotated checklist of extant brachyuran crabs of the world. Raffles B Zool 17:1–286
Falciai L (2002) First record of Menaethius monoceros (Latreille, 1825) (Decapoda, Majidae) in the central Tyrrhenian Sea. Crust. 75(10):1279–1283. https://doi.org/10.1163/156854002321518216
Sakai T (1969) Two new genera and twenty-two new species of crabs from Japan. Proc biol Soc Wash 82:243–280 text figs. 1–20, pls. 1–2
El-Sayed AA (1997) The biology of spider crab, Menaethius monoceros from the Red Sea and Gulf of Aqaba, Egypt. Egy J Aqua Biol Fish 1(1):1–16. https://doi.org/10.21608/EJABF.1997.3364
Sakai T (1976) Crabs of Japan and the adjacent seas: i-xxix, 1-773, 1-461, 1-16, maps 3, text figs. 1-379, pls. 1-251. (Kodansha, Tokyo)
Hendrickx ME (1995) Checklist of brachyuran crabs (Crustacea: Decapoda) from the eastern tropical Pacific. Med K Belg Inst Nat Wet. 65:125–150
Martin JW, Davis GE (2001) An updated classification of the recent Crustacea. Sci Ser Nat Hist Mus Los Angeles County 39(i–vii):1–124
McLaughlin PA, Camp DK, Angel MV et al (2005) Common and scientific names of aquatic invertebrates from the United States and Canada: crustaceans. Am. Fish Soc Sp Publ. 31:1–545
Guinot D (1966) Recherches préliminaires sur les groupements naturels chez les Crustacés Décapodes Brachyoures. I. Les affinités des genres Aethra, Osachila, Hepatus, Hepatella et Actaeomorpha. Bull Mus Natl d’Histoire Nat Paris 2(38(5)):744–762
Ng PKL (1998) Crabs. In: Carpenter KE, Niem VH (eds) FAO Species identification guide for fishery purposes. The living marine resources of the Western Central Pacific. Volume 2. Cephalopods, crustaceans, holothurians and sharks. Food and Agriculture Organisation, Rome, pp 1045–1155
Ng PKL, Wang CH, Ho PH, Shih HT (2001) An annotated checklist of brachyuran crabs from Taiwan (Crustacea: Decapoda), National Taiwan Museum Special Publication Series, vol 11, pp 1–86
Davie PJF (2002) Crustacea: Malacostraca. Eucarida (part 2). Decapoda – Anomura, Brachyura: In Zoological catalogue of Australia. (Eds A. Wells and W. W. K. Houston.) 19.3B. CSIRO Publishing, Melbourne. pp 641
Naderloo R (2017) Atlas of crabs of the Persian Gulf. Environment : Marine & Freshwater Sciences, Springer Nature, Germany
Head SM (1987) Introduction. In: Edwards AJ, Head SM (eds) Key environments: the Red Sea. Pergamon Press, Oxford
Por FD (1978) Lessepsian migration. The influx of Red Sea biota into the Mediterranean by way of the Suez Canal. Springer Verlag, Berlin
Heller C (1861) Synopsis der im Rothen Meere vorkommenden Crustaceen. Verhandlungen der Zoologisch-Botanischen Gesellschaft in Wien, vol 11, pp 3–32
Paulson O M (1875) Investigations on the Crustacea of the Red Sea with notes on Crustacea of the adjacent seas. Part. I. Podophthalmata and Edriophthalmata (Cumacea). Kiev, Kul’zhenko, i-xiv -t 1-144, pl. 1-21
Klunzinger CB (1906) Die Spitz- und Spitzmundkrabben (Oxyrhyncha und Oxystomata) des Roten Meeres, Stuttgart, vol 91, p VII
Nobili G (1906) Faune carcinologique de la Mer Rouge. Decapodes et Stomatopodes. Annales des Sciences Naturelles (Zoologie) 9e série. 4:1–347, figs. 1–12, pls. 1–11
Laurie RD (1915) On the Brachyura. Reports on the marine biology of the Sudanese Red Sea, from collections made by Cyril Crossland, M.A., B. Sc., F.Z.S., XXI. J Linnaean Soc (London) Zool 31(209):407–475. https://doi.org/10.1111/j.1096-3642.1915.tb00463.x
Balss H (1929) Expedition S. M. Schiff Pola in das Rote Meer. Nordliche und sudliche Halfte 1895/96-1897/98. Zool. Ergebn, 36. Decapoden des Roten Meeres, 4. Oxyrhyncha und Schlussbetrachtungen. Denkschr. Akad. Wiss. Wien (math-nat.), 102: 1–30
Guinot D (1962) Surune collection de Crustaces decapodes brachyoures de Mer Rouge et de Somalie. Boll Mus Civ Stor Nat Venezia 15:7–63
Guinot D (1962) Sur une collection de Crustaces decapodes brachyoures des iles Maldives et de Mer Rouge (Expedition Xarifa 1957-1958). Kieler Meeresforsch 18:231–244
Vine P (1986) Red Sea invertebrates. IMMEL publishing, London
Plaisance L, Knowlton N, Paulay G, Meyer C (2009) Reef-associated crustacean fauna: biodiversity estimates using semi-quantitative sampling and DNA barcoding. Coral Reefs. 28(4):977–986. https://doi.org/10.1007/s00338-009-0543-3
Hebert PDN, Cywinska A, Ball SL, DeWaard JR (2003) Biological identifications through DNA barcodes. Proc R Soc Lond. 270(1512):313–321. https://doi.org/10.1098/rspb.2002.2218
Costa FO, deWaard JR, Boutillier J, Ratnasingham S, Dooh RT, Hajibabaei M, Hebert PDN (2007) Biological identifications through DNA barcodes: the case of the Crustacea. Can J Fish Aquat Sci. 64(2):272–295. https://doi.org/10.1139/f07-008
Schubart CD, Neigle JE, Felder DL (2000) Use of the mitochondrial 16SrDNA gene for phylogenetic and population studies of Crustacea. Crust Issues 12:817–830
Kitaura J, Nishida M, Wada K (2006) The evolution of social behavior in sentinel crabs (Macrophthalmus): implications from molecular phylogeny. Biol J Linn Soc. 88(1):45–59. https://doi.org/10.1111/j.1095-8312.2006.00609.x
Macdonald KS, Rios R, Duffy JE (2006) Biodiversity, host specificity, and dominance by eusocial species among sponge-dwelling alpheid shrimp on the Belize Barrier Reef. Divers Distrib. 12(2):165–178. https://doi.org/10.1111/j.1366-9516.2005.00213.x
Porter ML, Cronin TW, McClellan DA, Crandall KA (2007) Molecular characterization of Crustacean visual pigments and the evolution of Pancrustacean opsins. Mol Biol Evol. 24(1):253–268. https://doi.org/10.1093/molbev/msl152
Lefébure T, Douady CJ, Gouy M, Gibert J (2006) Relationship between morphological taxonomy and molecular divergence within Crustacea: proposal of a molecular threshold to help species delimitation. Mol Phyl Evol. 40(2):435–447. https://doi.org/10.1016/j.ympev.2006.03.014
Sakai T (1938) Studies on the crabs of Japan, 3. Brachygnatha Oxyrhyncha: 193-364, figs. 1-55, pis. 20-41. (Yokendo Co., Tokyo)
Kumar S, Stecher G, Tamura K (2016) MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Mol Biol Evol. 33(7):1870–1874. https://doi.org/10.1093/molbev/msw054
Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol. 4(4):406–425. https://doi.org/10.1093/oxfordjournals.molbev.a040454
Tamura K (1992) Estimation of the number of nucleotide substitutions when there are strong transition-transversion and G + C-content biases. Mol Biol Evol. 9(4):678–687. https://doi.org/10.1093/oxfordjournals.molbev.a040752
Kimura M (1980) A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. J Mol Evol. 16(2):111–120. https://doi.org/10.1007/BF01731581
Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R (1994) DNA primers for amplification of mitochondrial cytochrome C oxidase subunit I from diverse metazoan invertebrates. Mol Mar Biol Biotechnol. 3(5):294–299
Mathews LM, Anker A (2009) Molecular phylogeny reveals extensive ancient and ongoing radiations in a snapping shrimp species complex (Crustacea, Alpheidae, Alpheus armillatus). Mol Phyl Evol. 50(2):268–281. https://doi.org/10.1016/j.ympev.2008.10.026
Zhang J, Kapli P, Pavlidis P, Stamatakis A (2013) A general species delimitation method with applications to phylogenetic placements. Bioinformatics. 29(22):2869–2876. https://doi.org/10.1093/bioinformatics/btt499
Bickford D, Lohman D, Sodhi N, Ng P, Meier R, Winker K, Ingram K (2007) Cryptic species as a window on diversity and conservation. Trends Ecol Evol. 22(3):148–155. https://doi.org/10.1016/j.tree.2006.11.004
Sites JW Jr, Marshall JC (2003) Delimiting species: a Renaissance issue in systematic biology, Trends Ecol. Evol. 18:462–470
Tautz D, Arctander P, Minelli A, Thomas RH, Vogler AP (2003) A plea for DNA taxonomy, Trends Ecol. Evol. 18:70–74
The author is very grateful to Professor Tohru Naruse and the management staff of Iriomote Station, Tropical Biosphere Research Center, and Professor James Davis Riemer, Faculty of Science, University of the Ryukyus, Okinawa, Japan, for hosting, helping, and providing all laboratory facilities used in the present study during the period February to August 2018. Also, the author is grateful to Professor Awaad A. M. El-Sayed for providing additional specimens, collected during the period from April 1996 to February 1997, and Egyptian Red Sea coasts for further morphological examination.
Ethics approval and consent to participate
Consent for publication
The author declares that there are no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
Amer, M.A. DNA barcoding of the spider crab Menaethius monoceros (Latreille, 1825) from the Red Sea, Egypt. J Genet Eng Biotechnol 19, 42 (2021). https://doi.org/10.1186/s43141-021-00141-2
- Red Sea crabs
- Horny crab