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First barcode of Ryphila cancellus (Herbst, 1783), from the southwest coast of India
Journal Pre-proof First barcode of Ryphila cancellus (Herbst, 1783), from the southwest coast of India Ajin Madhavan, Reshma Silvester, Prabhakaran M.P., Reza Naderloo, C.K. Radhakrishnan, N.R. Menon
PII: DOI: Reference:
S2352-4855(18)30591-7 https://doi.org/10.1016/j.rsma.2019.100910 RSMA 100910
To appear in:
Regional Studies in Marine Science
Received date : 15 November 2018 Revised date : 26 October 2019 Accepted date : 26 October 2019 Please cite this article as: A. Madhavan, R. Silvester, Prabhakaran M.P. et al., First barcode of Ryphila cancellus (Herbst, 1783), from the southwest coast of India. Regional Studies in Marine Science (2019), doi: https://doi.org/10.1016/j.rsma.2019.100910. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2019 Published by Elsevier B.V.
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First barcode of Ryphila cancellus (Herbst, 1783), from the southwest coast of
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India
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Ajin Madhavan1*, Reshma Silvester1, Prabhakaran M P2, Reza Naderloo3, C K
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Radhakrishnan1 and N R Menon4
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1. Department of Marine Biology, Microbiology and Biochemistry, School of Marine Sciences,
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Cochin University of Science and Technology, Lakeside Campus, Cochin – 682016, Kerala,
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India.
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2. Kerala University of Fisheries and Ocean Science, Panangad, Cochin- 682506, Kerala, India
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3. School of Biology, College of Science, University of Tehran, 14155-6455 Tehran, Iran. 4. Deceased March 17th 2018
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Corresponding author- Ajin Madhavan, email id: [email protected]
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Abstract
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Ryphila cancellus (Herbst, 1783), belonging to the family Leucosiidae has very unique
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morphological characteristics compared with the other crab families. They are distributed along the
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intertidal regions of Indo-pacific and prefer soft-bottom sandy beaches with gentle wave action.
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Due to very subtle morphological differences with other genera in the same family, identification
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based on the taxonomical examination alone is quite challenging. In the present study, a detailed
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morphological examination and DNA barcoding has been performed. The amplified mitochondrial
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CO1 gene of the crab specimens were sequenced and the obtained sequences were deposited in the
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Genbank. The neighbour-joining and distance matrix tree revealed the relationship between relative
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genera. The CO1 gene sequence divergence of Ryphila cancellus with other related groups ranged
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from 0.1712 to 0.9.
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Keywords: Ryphila cancellus; Sandy shore; Morphological identification; DNA barcoding;
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southwest coast
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1. Introduction
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The southwest coast of India is well known for its marked marine biodiversity. Intertidal ecosystems
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along the coast are major habitats for a wide variety of plant and animal communities. Sandy and
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rocky shores, muddy shores, elevated cliffs, estuaries and mangrove swamps are the major
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ecological habitats along the coast. Malacostracan crustaceans are one of the major groups of
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invertebrates inhabiting these coasts. These ecosystem engineers maintain and improve the
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sediment quality, help in remineralisation and also create a structurally suitable habitat for smaller
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vertebrates and invertebrates. Given their ecological and economic importance, efforts have been
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made for a detailed study of these animals. A total of 910 species of marine brachyuran crabs
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assigned to 361 genera and 62 families are listed from Indian waters (Trivedi et al., 2018). Among
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the crab families, Leucosiidae commonly called pebble crabs are very unique in their morphological
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features and contain three subfamilies (WoRMS, 2018). They are present in the intertidal, subtidal
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and also in deep waters of marine ecosystems. Most of the species were recorded from the subtidal
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and intertidal regions and are a diverse group in Indo-Pacific regions (Galil, 2009; Naderloo, 2017).
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A detailed examination of the genus Philyra leach, 1817 from the Leucosiidae family was done by
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Galil (2009), and confirmed that it is restricted to its type species, P. globus (Fabricius, 1775) and
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P. samia. Therefore, Galil (2009) assigned the genus Ryphila and described the type species as
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Ryphila cancellus (Herbst, 1783). Naderloo (2017) recorded 33 species of Leucosiidae from Persian
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Gulf and Gulf of Oman. From India, recently 6 species of leucosiid crabs including Ryphila
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cancellus were recorded from the Gujarat coastline (Beleem et al., 2014, 2017; Trivedi et al., 2016).
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Several species of crabs have been described from Indian waters based on their morphological
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characteristics. However, DNA barcoding for the identification of crab species has been lacking.
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Validation of the species based on DNA barcoding has been regarded as a very useful tool along
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with morphology-based identification (Teletchea, 2010). The primary goal of DNA barcoding is
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the assembly of reference sequence libraries derived from expert-identified voucher specimens in
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order to develop reliable molecular tools for the species identification in nature (Hubert et al., 2008).
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DNA barcoding also aids in identifying unfamiliar organisms, thus helping with biological surveys
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and conservation. Cryptic species that are morphologically similar are difficult to distinguish with
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the morphological examination alone. However, due to their genetically distinct character, DNA
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barcoding can be a very effective tool in the assessment of these cryptic species (Trivedi et al.,
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2016). There are several previous studies which have highlighted the importance of DNA barcoding
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as a species identification tool (Bucklin et al., 2007, 2009; Hebert et al., 2003; Hunt et al., 2010;
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Steinke et al., 2009; Valentini et al., 2009; Ward et al., 2005, 2008). Besides, barcoding helps non-
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experts to identify species in a rapid and cost- effective manner. Recently, DNA barcoding was
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carried out on a leucosiid crab, Lyphira perplexa from India (Sudharma et al., 2014). Even though
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there are few reports of Ryphila cancellus from the Indian coast, molecular characterisation of the
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species based on CO1 DNA barcoding was not previously undertaken and reports are lacking. In
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this present study, a detailed morphological examination of Ryphila cancellus was carried out. This
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is the pioneer study of CO1 barcoding of Ryphila cancellous.
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2. Material and methods
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Sample collection and morphological examination
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The specimens were collected during an intertidal survey from 4 different sandy beaches of Kerala,
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India. A total of 7 specimens RC1-RC7 (2 female and 5 males) were collected from different
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locations such as Payyambalam (11˚86’96’’N, 75˚35’21’’E), Muzhupilangadu (11˚79’63’’N,
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75˚44’19’’E), Varkala (8˚73’42’’N, 76˚70’79’’E) and Kovalam (8˚42’89’’N, 76˚95’81’’E) along
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the southwest coast of India. Specimens were handpicked and sometimes a shovel was used to dig
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out the animals. Specimens were closely observed in the field to understand their behaviour and
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feeding. Collected specimens were brought to the lab for further identification. Specimens preserved
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in 4% formalin were used for morphological identification. Stereo microscopes were used for a
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closer examination of the specimens and the identity was confirmed based on the previous
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description (Galil, 2009).
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DNA extraction, PCR amplification, sequencing and alignment
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Alcohol preserved (70%) crabs were rehydrated in 500 μl sterile distilled water for 10–12 h at room
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temperature (Bucklin et al., 1996 a, b,1995) prior to DNA extraction. Genomic DNA from the
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chelate legs of the crabs was extracted using the Marine animal DNA kit (Origin diagnostics) and
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the DNA was stored at -20 °C for further analysis. PCR amplification was performed in a total
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reaction volume of 25 μL using a gradient thermal cycler (Agilent SureCycler 8800, 2013). The
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primer pair LCO-1490 (5′-GGTCAACAAATCATAAAGATATTGG-3′) and HCO-2198 (5′-
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TAAACTTC AGGGTGACCAAAAAATCA-3′) were used to amplify mitochondrial CO1 gene
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sequences from the selected samples (Folmer et al., 1994). The PCR kit used was the Takara
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Clontech EmeraldAmp® GT PCR Master Mix (Takara Bio, Japan). The reaction mixture consisted
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of 12.5 μL PCR Master Mix, 1 μL each of forward and reverse primer, 4 μL template DNA and 6.5
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μL distilled water. The PCR reaction included an initial denaturation for 5 min at 94 °C, followed
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by 40 cycles of 1 min each at 94 °C for denaturation, 2 min at 37 °C for annealing and 3 min at 72
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°C for extension, with a final extension at 72 °C for 10 min. PCR products were then visualized on
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1.5 % agarose. Amplified products exhibiting intense bands after electrophoresis were sent to
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SciGenom Labs (SciGenom Labs Pvt, Ltd., Kerala, India) for sequencing.
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The obtained CO1 sequences of Ryphila cancellus were submitted to the Genbank. The CO1
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sequences of 11 closely related genera retrieved from the Genbank along with our sequences were
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exported to the BioEdit 7.0.9 and aligned using the ClustalW and the genetic distance was
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determined using MEGA6 software.
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3. Results
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Systematic accounts
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Ryphila cancellus (Herbst, 1783)
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Order Decapoda Latreille, 1802
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Genus Ryphila, Galil, 2009
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Family Leucosiidae; Samouelle, 1819
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Cancer cancellus, Herbst 1783: 94, 95, pl 2, fig 20.
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Leucosia scabriuscula, Weber 1795: 92; Fabricius, 1798: 349; Lichtenstein 1816: 142; Latreille
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1802: 116; Bosc 1802: 237; 1830: 288. Zimsen 1964: 651.
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Philyra scabriuscula, Alcock 1896: 238 (in key), 239, 240; Ihle 1918: 275, 315, figs 97, 102, 115;
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Stephensen 1946: 88, 89, fig 15c–e; Guinot 1967a: 249 (in list); Hogarth 1989: 115; Tirmizi and
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Kazmi 1988: 98 (in key), 106–109, fig 31a–i; Hornby 1997: 16.
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Philyra cancella , Sakai 1999: 17, 18, pl 7b.
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Ryphila cancellus,Galil 2009: 310–312, figs 21a, 22; Naderloo and Türkay 2012: 32; Naderloo et
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al. 2013: 449, tab 1, 458, tab 3.
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Materials examined: Collected specimens RC1♀- 9.3 mm, RC2♀-10.6 mm, RC3♂-12.4 mm,
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RC4♂-11.2 mm, RC5♂- 13.4, RC6♂ -12.8 mm, RC7♂-11.9mm, from the sandy intertidal zones of
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Payyambalam (11˚86′96″N, 75˚35′21″E), Muzhupilangadu (11˚79′63″N, 75˚44′19″E), Varkala
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(8˚73′42″N, 76˚70′79″E) and Kovalam (8˚42′89″N, 76˚95′81″E).
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Description: Adult
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The carapace is discoid or oval with serrated and granulated margins (Figure 1). The anterior region
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of the carapace is slightly produced. Closely spaced granules are present on the dorsal surface of
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the carapace conspicuously visible along the anterior, posterior and lateral margins. Granules on the
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anterior margin of carapace larger than granules on posterolateral and posterior margins. Shallow
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branchio cardiac and cervical grooves are present on the dorsal side of the carapace. The branchial
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region is bulbous. There are no lateral spines. The frontal region is slightly projected and medially
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grooved. The rostrum is very small. Short antennae are inserted between the antennular fossa and
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orbit. It has small eyes and orbits. Epistome is continuous with expanded margins, projecting
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beyond the frontal margin. Chelipeds are longer and subequal. Cheliped merus is subcylindrical and
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slightly longer than the carapace in males. The dorsal surface of the merus is marked with distinct
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parallel rows of shining granules which are decreasing in size and diminishing distally. Chela is flat
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and slightly broader than the merus (Figure 1). Minute granules are present along the inner margins
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of the carpus and upper and lower surfaces of the propodus (Figure 2). Inner margins of the dactylus
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and pollex are serrulated with more prominent serrulations distally. Pereiopods are slender and short
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with a subcylindirical meri which is longer than carpi and propodi. Upper and lower propodal
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margins are carinate. Dactyli is longer than propodi which is lanceolate and terminating in cornuted
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tips (Figure 1).
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Female: Abdomen is discoidal and covering most of the ventral surface. Fused abdominal segments
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are laterally granulated in first and second abdominal segments. First two female abdominal
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segments are transversely narrow. Segments three to six are fused, greatly enlarged and is like a
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shield. Telson is discoidal (Figure 2).
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Male: Male first pleopod with apical process filiform, needle-like and is nearly as long as shaft.
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Male abdominal sulcus is deep and nearly reaching the buccal cavity. First and second abdominal
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segments are transverse, narrow and swollen laterally. Third to sixth segments are fused and have
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indistinct suture lines with lateral margins bearing three indistinct ridges fitting into sutures between
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sternal segments. Telson is elongate and laciniate (Figure 1).
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Colour: The upper surface of the carapace and the anterior portion of the legs are more or less dark
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gray colour marbled with a light violet colour; the ventral side of the carapace and legs are pale rose
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or white in colour. Granules on the body are of white colour.
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Distribution and habitat
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Ryphila cancellus is distributed along the intertidal and sublittoral sandy shores of the Indo-pacific
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region. So far they have been reported from East Africa, Persian Gulf, Gulf of Oman, Arabian sea,
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Andaman Islands and east and west coasts of India and Australia (Galil, 2009). From India, they
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were previously recorded from Tamilnadu, Maharashtra, Gujarat and Goa (Beleem et al., 2014,
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2017; Galil, 2009; Trivedi et al., 2016). Their abundance was more on the sandy shores with very
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gentle slopes and moderate wave action. By observation, it is confirmed that these crabs prefer
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shaded and protected shallow over unshaded and exposed sandy shore habitats. The nature of
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substrata they have selected for their habitat was wet, soft, fine and loose sand grains (Table 1). The
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abundance was comparatively higher in Muzhupilangadu, which is a dissipative type of beach with
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soft and fine substratum along the southwest coast of India (Table 1). This beach was characterized
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by its sheltered nature and wave disturbance was low. So this indicates that these crabs prefer this
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habitat over other types of beaches. Their presence was also noted from substrates with coarser
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particles but was rare. However, a recent study reported their occurrence from the crevices of upper
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intertidal rock (Beleem et al., 2017). The pale colouration and granulated body of the crab allow it
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to blend in with its surroundings and its colouration varies slightly depending on the environment.
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Unlike most crabs, they don’t make a permanent burrow but are capable of hiding beneath the sand
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with the help of their legs and is easily done when they are submerged in shallow water. In general,
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these crabs are delicate and soft and their long chelipeds help them to catch smaller prey. Based on
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the observation it is understood that they feed on small crustaceans and other invertebrates such as
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amphipods, mole crabs and polychaetes. Their breeding was observed during the late-monsoon and
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the juveniles were found during the post-monsoon season.
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Remarks
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Ryphila cancellus is a common species of the intertidal sandy shores along the Indo-pacific region.
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There has been a recent description of the species from the Gulf of Oman and also from, Gujarat
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coast, India (Beleem et al., 2017; Naderloo, 2017). The present study again confirmed the identity
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of the crab and the morphological diagnosis stands close to the observation of previous descriptions
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(Galil, 2009; Beleem et al., 2017; Naderloo, 2017). However, the examined specimen showed
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notable differences in their colour and patterns over previously described ones. Colour patterns were
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more distinct and moderately darker and showed more similarities with the specimen described by
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Naderloo, 2017. Light grey shades on the chelipeds were more visible over the pale violet colour.
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Marginal difference in the shape of the carapace was observed when compared with the specimen
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described by Beleem et al., 2017. Carapace was more discoidal, cervical grove more prominent in
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the present specimen. The frontal margin was more protruded in the specimen described by Beleem
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et al., 2017. However, specimens showed very close similarities with the previous ones described
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by Galil (2009) and Naderloo (2017).
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DNA barcoding and phylogenetic analysis
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The similarity of the newly obtained mitochondrial CO1 gene sequences was checked using the
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NCBI nucleotide BLAST tool. The sequences exhibited a maximum of 84% similarity only with
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the existing CO1 sequences in the Genbank. Since the previous record of Ryphila cancellus is not
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available in the Genbank database, we report this as a pioneer DNA barcode of Ryphila cancellus.
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The sequences were submitted to the Genbank (NCBI) under the accession numbers KT907442,
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KT907443 and KT907444.
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The obtained DNA sequences were aligned with CO1 sequences of other crabs retrieved from
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Genbank. Eleven CO1 sequences from the members of genera Caphyra, Tyche, Cyclodius, Pilodius,
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Lyphira, Phylira, Percephona, Iliacantha and Portunus were incorporated for constructing the
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neighbour-joining tree. The tree is drawn to scale, with branch length in the same units as those of
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the distances used to infer the tree. The phylogram clearly exhibits the relationship of Ryphila
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cancellus with other related genera (Figure 3). Portunus pelagicus KJ701524 formed a separate
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cluster. These species revealed a divergent array in the tree (Figure 3). A distance matrix tree
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revealed divergence of Ryphila from the other related genera (Figure 4). The number of base
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substitutions per site between sequences are shown (Figure 4). Analyses were conducted using the
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Kimura 2-parameter model. The analysis involved 14 nucleotide sequences.
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The sequence divergence of genus Ryphila with other selected individuals in the group ranged from
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0.1712 to 0.9. Portunus pelagicus KJ701524 was the most distant relative of R. cancellus (Herbst,
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1783), with a genetic distance of 0.9.
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4. Discussion
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The significance of biological collection with distinct and detailed morphological diagnosis has
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increased in recent years.The combination of taxonomic and molecular marker-based studies
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provides a strong support for future research. Therefore, the present study focused on the
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morphological diagnosis combined with DNA barcoding of Ryphila cancellus. Ryphila cancellus
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has the widest distribution in the Indo-West Pacific: East Africa, Madagascar, southern Oman,
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Persian Gulf, Gulf of Oman, Pakistan, India, Mergui Archipelago, Malay Archipelago, Burma,
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Thailand, Borneo, Sumatra and Australia (Naderloo, 2017). Besides, the present study provides
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additional information regarding the distribution, habitat and substrate preference of this species.
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They are found to inhabit the soft bottom sediment of the intertidal region, hiding submerged in the
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sediment with the help of their hind periopods. In general, they prefer to live in dissipative type of
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sandy beaches with moderate wave action and remain hidden under the wet sand without exposing
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themselves.
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Until the detailed examination of genus Philyra (Leach, 1817) by Galil in 2009, this species was
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mistaken for many of its closely related species. Herbst in 1783 reported this species as Cancer
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cancellus (Herbst, 1783) and later Fabricius recognised it as Leucosia scabriuscula (Fabricius,
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1798). Leach assigned the species as Philyra scabriuscula (Leach, 1817). Detailed examination of
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the genus Philyra (1817) revealed the exact identity of this animal and assigned it to a new genus
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Ryphila (Galil, 2009). The similarities of the species (eg. shape of the carapace, distribution of
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granules, size, width, length and morphology of the chelipids, habitat) with other closely related
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genera of the leucosiidae family itself made it difficult to differentiate these animals. It is noted that
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Ryphila verrucosa (Henderson, 1893), a closely related species of Ryphila cancellus, can be
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distinguished with the presence of its entirely granulose dorsal surface of the carapace and thoracic
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sternites. Hence, the present study was undertaken to solve this taxonomic ambiguity of the species
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with the help of molecular markers along with a clear and detailed morphological examination. The
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present species was compared with the description of Galil (2009), which is accounted to be the
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most reliable one. Only marginal differences were noticed in the morphology of the present Ryphila
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cancellus specimen on comparison with the details specified by Galil. The morphological
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differences pertained to colouration, size and distribution of granule on the carapace, and chelipids.
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It is assumed that these are only ecotypical variations. The use of molecular markers has become
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very relevant to elucidate the phylogenetic features of marine invertebrates. The CO1 gene is highly
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conserved and evolves rather slowly when compared with other protein coded mitochondrial genes,
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which has resulted in employing this as a potential molecular marker in phylogenetic studies (Russo
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et al., 1996). The CO1 amplicons obtained from three (RC1-RC3) specimens were deposited in the
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Genbank. In the present study, the phylogram constructed by the neighbour-joining method using
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the COI barcoding gene suggested a clear separation between Ryphila cancellus and the other
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closely related genera. Apart from their similar morphological characteristics with the closely
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related genera, they are completely outgrouped in the phylogenetic tree. From the distance matrix
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data, genetic distance between the selected individuals were clearly evident. Even though Phylira
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and Ryphila shared almost similar morphological features, they exhibited greater genetic divergence
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and exhibited a genetic distance of 0.7645.
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Different species assigned to various genera of Leucosiidae are known to inhabit sub-tidal and the
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shelf regions of the Indo-Australian archipelago. Several related species are described from this area
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and hence it would be worthwhile to conduct DNA barcoding in these species as well. In this
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respect, the presence of the specimen belonging to Ryphila cancellus in the intertidal area is
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significant. Since this species was wrongly assigned to various other genera by previous authors,
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the COI barcoding would solve the taxonomic ambiguity of this genus.
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Acknowledgement
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The authors would like to thank the Head, Department of Marine Biology, Microbiology and
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Biochemistry and Nansen Environmental Research Centre (India) for the financial support and
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facilities provided. Sequencing facility provided by Scigenom labs, Kerala, India is also greatly
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acknowledged.
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Conflict of interest
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The authors declare that there are no potential conflicts of interest.
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Venkataraman, K., Wafar, M., 2005. Coastal and Marine Diversity of India. IJMS 34, 57-75.
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Ward, R. D., B. H. Holmes and T. D. O’Hara. 2008. DNA barcoding discriminates echinoderm
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species. Mol Ecol Res 8, 1202-1211. https://doi: 10.1111/j.1755-0998.2008.02332.x Ward, R. D., Zemlak, T. S., Bronwyn, H. I., Last, P. R., Hebert, P. D. N., 2005. DNA barcoding Australia’s
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Weber, F., 1795. Nomenclator entomologicus secundum Entomologiam systematicam ill. Fabricii adjectis speciebus recens detectis et varietatibus. Viii+171 pp. Chilonii & Hamburgi. WoRMS
(2019).
Leucosiidae
Samouelle,
1819.
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at:
http://www.marinespecies.org/aphia.php?p=taxdetails&id=106758 on 2019-03-04
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Zimsen, E. 1964. The Type Material of I. C. Fabricius. Munksgaard, Copenhagen, 656 p., 1 text-fi
375 376
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g., 2.
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Figure captions
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Table 1: The habitat and abundance of Ryphila cancellus (Herbst, 1783).
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Figure 1: Ryphila cancellus, (Herbst, 1783) -Dorsal View (Male).
381
Figure 2: Ryphila cancellus, (Herbst, 1783)- Ventral view of male (a) and female (b).
382
Figure 3: Phylogenetic tree exhibiting the evolutionary relationship of Ryphila cancellus (Herbst,
383
1783) with other related genera.
384
Figure 4: Distance matrix tree displaying inter-generic divergence of Ryphila cancellus (Herbst,
385
1783) with closely related genera. The number of base substitutions per site between sequences
386
are shown. Analyses were conducted using the Kimura 2-parameter model.
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391 392 393 394
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Table 1 Location
Latitude and longitude
Beach
Zone
Type of substratum
Payyambalam
11˚86’96’’N, 75˚35’21’’E
SandyIntermediate
Intertidal (lower littoral)
Coarse and loose sand
SandyDissipative
Intertidal (Mid-littoral and lower littoral ) Intertidal (Mid-littoral)
Fine and soft sediment
1
Specimens (number of specimens collected) 1
2
3
Muzhupilangadu 11˚79’63’’N, 75˚44’19’’E
3
1
Varkala
8˚73’42’’N, 76˚70’79’’E
4
2
Kovalam
8˚42’89’’N, 76˚95’81’’E
396
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397 398
402 403 404 405 406
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399 400
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Sl. No
SandyIntermediate
Sandy and rocky patchesIntermediate
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395
Intertidal (Mid-littoral)
Coarse (medium) sand Coarse (medium)
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Figure 1
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Figure 2
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420
Figure 3
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Figure 4
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Highlights
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The paper reports the DNA barcoding of crab Ryphila cancellus (Herbst, 1783)
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Conducted taxonomic and molecular identification of the crab specimens
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The CO1 gene of the crab was sequenced
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The neighbor joining tree and distance matrix were plotted
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Sequences were deposited in the Genbank as first molecular data of the species
435 436
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Conflict of interest declaration
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The authors declare there are no potential conflict of interest in the following
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manuscript titled “First barcode of Ryphila cancellus (Herbst, 1783), from the
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southwest coast of India”
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PII: DOI: Reference:
S2352-4855(18)30591-7 https://doi.org/10.1016/j.rsma.2019.100910 RSMA 100910
To appear in:
Regional Studies in Marine Science
Received date : 15 November 2018 Revised date : 26 October 2019 Accepted date : 26 October 2019 Please cite this article as: A. Madhavan, R. Silvester, Prabhakaran M.P. et al., First barcode of Ryphila cancellus (Herbst, 1783), from the southwest coast of India. Regional Studies in Marine Science (2019), doi: https://doi.org/10.1016/j.rsma.2019.100910. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2019 Published by Elsevier B.V.
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First barcode of Ryphila cancellus (Herbst, 1783), from the southwest coast of
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India
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Ajin Madhavan1*, Reshma Silvester1, Prabhakaran M P2, Reza Naderloo3, C K
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Radhakrishnan1 and N R Menon4
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1. Department of Marine Biology, Microbiology and Biochemistry, School of Marine Sciences,
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Cochin University of Science and Technology, Lakeside Campus, Cochin – 682016, Kerala,
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India.
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2. Kerala University of Fisheries and Ocean Science, Panangad, Cochin- 682506, Kerala, India
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3. School of Biology, College of Science, University of Tehran, 14155-6455 Tehran, Iran. 4. Deceased March 17th 2018
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Corresponding author- Ajin Madhavan, email id: [email protected]
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Abstract
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Ryphila cancellus (Herbst, 1783), belonging to the family Leucosiidae has very unique
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morphological characteristics compared with the other crab families. They are distributed along the
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intertidal regions of Indo-pacific and prefer soft-bottom sandy beaches with gentle wave action.
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Due to very subtle morphological differences with other genera in the same family, identification
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based on the taxonomical examination alone is quite challenging. In the present study, a detailed
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morphological examination and DNA barcoding has been performed. The amplified mitochondrial
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CO1 gene of the crab specimens were sequenced and the obtained sequences were deposited in the
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Genbank. The neighbour-joining and distance matrix tree revealed the relationship between relative
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genera. The CO1 gene sequence divergence of Ryphila cancellus with other related groups ranged
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from 0.1712 to 0.9.
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Keywords: Ryphila cancellus; Sandy shore; Morphological identification; DNA barcoding;
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southwest coast
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1. Introduction
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The southwest coast of India is well known for its marked marine biodiversity. Intertidal ecosystems
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along the coast are major habitats for a wide variety of plant and animal communities. Sandy and
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rocky shores, muddy shores, elevated cliffs, estuaries and mangrove swamps are the major
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ecological habitats along the coast. Malacostracan crustaceans are one of the major groups of
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invertebrates inhabiting these coasts. These ecosystem engineers maintain and improve the
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sediment quality, help in remineralisation and also create a structurally suitable habitat for smaller
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vertebrates and invertebrates. Given their ecological and economic importance, efforts have been
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made for a detailed study of these animals. A total of 910 species of marine brachyuran crabs
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assigned to 361 genera and 62 families are listed from Indian waters (Trivedi et al., 2018). Among
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the crab families, Leucosiidae commonly called pebble crabs are very unique in their morphological
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features and contain three subfamilies (WoRMS, 2018). They are present in the intertidal, subtidal
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and also in deep waters of marine ecosystems. Most of the species were recorded from the subtidal
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and intertidal regions and are a diverse group in Indo-Pacific regions (Galil, 2009; Naderloo, 2017).
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A detailed examination of the genus Philyra leach, 1817 from the Leucosiidae family was done by
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Galil (2009), and confirmed that it is restricted to its type species, P. globus (Fabricius, 1775) and
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P. samia. Therefore, Galil (2009) assigned the genus Ryphila and described the type species as
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Ryphila cancellus (Herbst, 1783). Naderloo (2017) recorded 33 species of Leucosiidae from Persian
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Gulf and Gulf of Oman. From India, recently 6 species of leucosiid crabs including Ryphila
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cancellus were recorded from the Gujarat coastline (Beleem et al., 2014, 2017; Trivedi et al., 2016).
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Several species of crabs have been described from Indian waters based on their morphological
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characteristics. However, DNA barcoding for the identification of crab species has been lacking.
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Validation of the species based on DNA barcoding has been regarded as a very useful tool along
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with morphology-based identification (Teletchea, 2010). The primary goal of DNA barcoding is
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the assembly of reference sequence libraries derived from expert-identified voucher specimens in
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order to develop reliable molecular tools for the species identification in nature (Hubert et al., 2008).
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DNA barcoding also aids in identifying unfamiliar organisms, thus helping with biological surveys
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and conservation. Cryptic species that are morphologically similar are difficult to distinguish with
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the morphological examination alone. However, due to their genetically distinct character, DNA
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barcoding can be a very effective tool in the assessment of these cryptic species (Trivedi et al.,
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2016). There are several previous studies which have highlighted the importance of DNA barcoding
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as a species identification tool (Bucklin et al., 2007, 2009; Hebert et al., 2003; Hunt et al., 2010;
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Steinke et al., 2009; Valentini et al., 2009; Ward et al., 2005, 2008). Besides, barcoding helps non-
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experts to identify species in a rapid and cost- effective manner. Recently, DNA barcoding was
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carried out on a leucosiid crab, Lyphira perplexa from India (Sudharma et al., 2014). Even though
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there are few reports of Ryphila cancellus from the Indian coast, molecular characterisation of the
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species based on CO1 DNA barcoding was not previously undertaken and reports are lacking. In
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this present study, a detailed morphological examination of Ryphila cancellus was carried out. This
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is the pioneer study of CO1 barcoding of Ryphila cancellous.
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2. Material and methods
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Sample collection and morphological examination
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The specimens were collected during an intertidal survey from 4 different sandy beaches of Kerala,
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India. A total of 7 specimens RC1-RC7 (2 female and 5 males) were collected from different
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locations such as Payyambalam (11˚86’96’’N, 75˚35’21’’E), Muzhupilangadu (11˚79’63’’N,
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75˚44’19’’E), Varkala (8˚73’42’’N, 76˚70’79’’E) and Kovalam (8˚42’89’’N, 76˚95’81’’E) along
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the southwest coast of India. Specimens were handpicked and sometimes a shovel was used to dig
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out the animals. Specimens were closely observed in the field to understand their behaviour and
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feeding. Collected specimens were brought to the lab for further identification. Specimens preserved
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in 4% formalin were used for morphological identification. Stereo microscopes were used for a
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closer examination of the specimens and the identity was confirmed based on the previous
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description (Galil, 2009).
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DNA extraction, PCR amplification, sequencing and alignment
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Alcohol preserved (70%) crabs were rehydrated in 500 μl sterile distilled water for 10–12 h at room
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temperature (Bucklin et al., 1996 a, b,1995) prior to DNA extraction. Genomic DNA from the
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chelate legs of the crabs was extracted using the Marine animal DNA kit (Origin diagnostics) and
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the DNA was stored at -20 °C for further analysis. PCR amplification was performed in a total
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reaction volume of 25 μL using a gradient thermal cycler (Agilent SureCycler 8800, 2013). The
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primer pair LCO-1490 (5′-GGTCAACAAATCATAAAGATATTGG-3′) and HCO-2198 (5′-
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TAAACTTC AGGGTGACCAAAAAATCA-3′) were used to amplify mitochondrial CO1 gene
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sequences from the selected samples (Folmer et al., 1994). The PCR kit used was the Takara
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Clontech EmeraldAmp® GT PCR Master Mix (Takara Bio, Japan). The reaction mixture consisted
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of 12.5 μL PCR Master Mix, 1 μL each of forward and reverse primer, 4 μL template DNA and 6.5
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μL distilled water. The PCR reaction included an initial denaturation for 5 min at 94 °C, followed
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by 40 cycles of 1 min each at 94 °C for denaturation, 2 min at 37 °C for annealing and 3 min at 72
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°C for extension, with a final extension at 72 °C for 10 min. PCR products were then visualized on
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1.5 % agarose. Amplified products exhibiting intense bands after electrophoresis were sent to
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SciGenom Labs (SciGenom Labs Pvt, Ltd., Kerala, India) for sequencing.
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The obtained CO1 sequences of Ryphila cancellus were submitted to the Genbank. The CO1
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sequences of 11 closely related genera retrieved from the Genbank along with our sequences were
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exported to the BioEdit 7.0.9 and aligned using the ClustalW and the genetic distance was
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determined using MEGA6 software.
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3. Results
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Systematic accounts
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Ryphila cancellus (Herbst, 1783)
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Order Decapoda Latreille, 1802
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Genus Ryphila, Galil, 2009
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Family Leucosiidae; Samouelle, 1819
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Cancer cancellus, Herbst 1783: 94, 95, pl 2, fig 20.
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Leucosia scabriuscula, Weber 1795: 92; Fabricius, 1798: 349; Lichtenstein 1816: 142; Latreille
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1802: 116; Bosc 1802: 237; 1830: 288. Zimsen 1964: 651.
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Philyra scabriuscula, Alcock 1896: 238 (in key), 239, 240; Ihle 1918: 275, 315, figs 97, 102, 115;
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Stephensen 1946: 88, 89, fig 15c–e; Guinot 1967a: 249 (in list); Hogarth 1989: 115; Tirmizi and
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Kazmi 1988: 98 (in key), 106–109, fig 31a–i; Hornby 1997: 16.
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Philyra cancella , Sakai 1999: 17, 18, pl 7b.
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Ryphila cancellus,Galil 2009: 310–312, figs 21a, 22; Naderloo and Türkay 2012: 32; Naderloo et
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al. 2013: 449, tab 1, 458, tab 3.
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Materials examined: Collected specimens RC1♀- 9.3 mm, RC2♀-10.6 mm, RC3♂-12.4 mm,
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RC4♂-11.2 mm, RC5♂- 13.4, RC6♂ -12.8 mm, RC7♂-11.9mm, from the sandy intertidal zones of
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Payyambalam (11˚86′96″N, 75˚35′21″E), Muzhupilangadu (11˚79′63″N, 75˚44′19″E), Varkala
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(8˚73′42″N, 76˚70′79″E) and Kovalam (8˚42′89″N, 76˚95′81″E).
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Description: Adult
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The carapace is discoid or oval with serrated and granulated margins (Figure 1). The anterior region
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of the carapace is slightly produced. Closely spaced granules are present on the dorsal surface of
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the carapace conspicuously visible along the anterior, posterior and lateral margins. Granules on the
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anterior margin of carapace larger than granules on posterolateral and posterior margins. Shallow
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branchio cardiac and cervical grooves are present on the dorsal side of the carapace. The branchial
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region is bulbous. There are no lateral spines. The frontal region is slightly projected and medially
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grooved. The rostrum is very small. Short antennae are inserted between the antennular fossa and
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orbit. It has small eyes and orbits. Epistome is continuous with expanded margins, projecting
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beyond the frontal margin. Chelipeds are longer and subequal. Cheliped merus is subcylindrical and
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slightly longer than the carapace in males. The dorsal surface of the merus is marked with distinct
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parallel rows of shining granules which are decreasing in size and diminishing distally. Chela is flat
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and slightly broader than the merus (Figure 1). Minute granules are present along the inner margins
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of the carpus and upper and lower surfaces of the propodus (Figure 2). Inner margins of the dactylus
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and pollex are serrulated with more prominent serrulations distally. Pereiopods are slender and short
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with a subcylindirical meri which is longer than carpi and propodi. Upper and lower propodal
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margins are carinate. Dactyli is longer than propodi which is lanceolate and terminating in cornuted
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tips (Figure 1).
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Female: Abdomen is discoidal and covering most of the ventral surface. Fused abdominal segments
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are laterally granulated in first and second abdominal segments. First two female abdominal
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segments are transversely narrow. Segments three to six are fused, greatly enlarged and is like a
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shield. Telson is discoidal (Figure 2).
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Male: Male first pleopod with apical process filiform, needle-like and is nearly as long as shaft.
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Male abdominal sulcus is deep and nearly reaching the buccal cavity. First and second abdominal
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segments are transverse, narrow and swollen laterally. Third to sixth segments are fused and have
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indistinct suture lines with lateral margins bearing three indistinct ridges fitting into sutures between
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sternal segments. Telson is elongate and laciniate (Figure 1).
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Colour: The upper surface of the carapace and the anterior portion of the legs are more or less dark
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gray colour marbled with a light violet colour; the ventral side of the carapace and legs are pale rose
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or white in colour. Granules on the body are of white colour.
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Distribution and habitat
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Ryphila cancellus is distributed along the intertidal and sublittoral sandy shores of the Indo-pacific
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region. So far they have been reported from East Africa, Persian Gulf, Gulf of Oman, Arabian sea,
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Andaman Islands and east and west coasts of India and Australia (Galil, 2009). From India, they
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were previously recorded from Tamilnadu, Maharashtra, Gujarat and Goa (Beleem et al., 2014,
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2017; Galil, 2009; Trivedi et al., 2016). Their abundance was more on the sandy shores with very
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gentle slopes and moderate wave action. By observation, it is confirmed that these crabs prefer
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shaded and protected shallow over unshaded and exposed sandy shore habitats. The nature of
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substrata they have selected for their habitat was wet, soft, fine and loose sand grains (Table 1). The
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abundance was comparatively higher in Muzhupilangadu, which is a dissipative type of beach with
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soft and fine substratum along the southwest coast of India (Table 1). This beach was characterized
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by its sheltered nature and wave disturbance was low. So this indicates that these crabs prefer this
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habitat over other types of beaches. Their presence was also noted from substrates with coarser
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particles but was rare. However, a recent study reported their occurrence from the crevices of upper
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intertidal rock (Beleem et al., 2017). The pale colouration and granulated body of the crab allow it
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to blend in with its surroundings and its colouration varies slightly depending on the environment.
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Unlike most crabs, they don’t make a permanent burrow but are capable of hiding beneath the sand
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with the help of their legs and is easily done when they are submerged in shallow water. In general,
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these crabs are delicate and soft and their long chelipeds help them to catch smaller prey. Based on
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the observation it is understood that they feed on small crustaceans and other invertebrates such as
166
amphipods, mole crabs and polychaetes. Their breeding was observed during the late-monsoon and
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the juveniles were found during the post-monsoon season.
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Remarks
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Ryphila cancellus is a common species of the intertidal sandy shores along the Indo-pacific region.
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There has been a recent description of the species from the Gulf of Oman and also from, Gujarat
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coast, India (Beleem et al., 2017; Naderloo, 2017). The present study again confirmed the identity
172
of the crab and the morphological diagnosis stands close to the observation of previous descriptions
173
(Galil, 2009; Beleem et al., 2017; Naderloo, 2017). However, the examined specimen showed
174
notable differences in their colour and patterns over previously described ones. Colour patterns were
175
more distinct and moderately darker and showed more similarities with the specimen described by
176
Naderloo, 2017. Light grey shades on the chelipeds were more visible over the pale violet colour.
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Marginal difference in the shape of the carapace was observed when compared with the specimen
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described by Beleem et al., 2017. Carapace was more discoidal, cervical grove more prominent in
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the present specimen. The frontal margin was more protruded in the specimen described by Beleem
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et al., 2017. However, specimens showed very close similarities with the previous ones described
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by Galil (2009) and Naderloo (2017).
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DNA barcoding and phylogenetic analysis
183
The similarity of the newly obtained mitochondrial CO1 gene sequences was checked using the
184
NCBI nucleotide BLAST tool. The sequences exhibited a maximum of 84% similarity only with
185
the existing CO1 sequences in the Genbank. Since the previous record of Ryphila cancellus is not
186
available in the Genbank database, we report this as a pioneer DNA barcode of Ryphila cancellus.
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The sequences were submitted to the Genbank (NCBI) under the accession numbers KT907442,
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KT907443 and KT907444.
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The obtained DNA sequences were aligned with CO1 sequences of other crabs retrieved from
190
Genbank. Eleven CO1 sequences from the members of genera Caphyra, Tyche, Cyclodius, Pilodius,
191
Lyphira, Phylira, Percephona, Iliacantha and Portunus were incorporated for constructing the
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neighbour-joining tree. The tree is drawn to scale, with branch length in the same units as those of
193
the distances used to infer the tree. The phylogram clearly exhibits the relationship of Ryphila
194
cancellus with other related genera (Figure 3). Portunus pelagicus KJ701524 formed a separate
195
cluster. These species revealed a divergent array in the tree (Figure 3). A distance matrix tree
196
revealed divergence of Ryphila from the other related genera (Figure 4). The number of base
197
substitutions per site between sequences are shown (Figure 4). Analyses were conducted using the
198
Kimura 2-parameter model. The analysis involved 14 nucleotide sequences.
199
The sequence divergence of genus Ryphila with other selected individuals in the group ranged from
200
0.1712 to 0.9. Portunus pelagicus KJ701524 was the most distant relative of R. cancellus (Herbst,
201
1783), with a genetic distance of 0.9.
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4. Discussion
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The significance of biological collection with distinct and detailed morphological diagnosis has
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increased in recent years.The combination of taxonomic and molecular marker-based studies
205
provides a strong support for future research. Therefore, the present study focused on the
206
morphological diagnosis combined with DNA barcoding of Ryphila cancellus. Ryphila cancellus
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has the widest distribution in the Indo-West Pacific: East Africa, Madagascar, southern Oman,
208
Persian Gulf, Gulf of Oman, Pakistan, India, Mergui Archipelago, Malay Archipelago, Burma,
209
Thailand, Borneo, Sumatra and Australia (Naderloo, 2017). Besides, the present study provides
210
additional information regarding the distribution, habitat and substrate preference of this species.
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They are found to inhabit the soft bottom sediment of the intertidal region, hiding submerged in the
212
sediment with the help of their hind periopods. In general, they prefer to live in dissipative type of
213
sandy beaches with moderate wave action and remain hidden under the wet sand without exposing
214
themselves.
215
Until the detailed examination of genus Philyra (Leach, 1817) by Galil in 2009, this species was
216
mistaken for many of its closely related species. Herbst in 1783 reported this species as Cancer
217
cancellus (Herbst, 1783) and later Fabricius recognised it as Leucosia scabriuscula (Fabricius,
218
1798). Leach assigned the species as Philyra scabriuscula (Leach, 1817). Detailed examination of
219
the genus Philyra (1817) revealed the exact identity of this animal and assigned it to a new genus
220
Ryphila (Galil, 2009). The similarities of the species (eg. shape of the carapace, distribution of
221
granules, size, width, length and morphology of the chelipids, habitat) with other closely related
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genera of the leucosiidae family itself made it difficult to differentiate these animals. It is noted that
223
Ryphila verrucosa (Henderson, 1893), a closely related species of Ryphila cancellus, can be
224
distinguished with the presence of its entirely granulose dorsal surface of the carapace and thoracic
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sternites. Hence, the present study was undertaken to solve this taxonomic ambiguity of the species
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with the help of molecular markers along with a clear and detailed morphological examination. The
227
present species was compared with the description of Galil (2009), which is accounted to be the
228
most reliable one. Only marginal differences were noticed in the morphology of the present Ryphila
229
cancellus specimen on comparison with the details specified by Galil. The morphological
230
differences pertained to colouration, size and distribution of granule on the carapace, and chelipids.
231
It is assumed that these are only ecotypical variations. The use of molecular markers has become
232
very relevant to elucidate the phylogenetic features of marine invertebrates. The CO1 gene is highly
233
conserved and evolves rather slowly when compared with other protein coded mitochondrial genes,
234
which has resulted in employing this as a potential molecular marker in phylogenetic studies (Russo
235
et al., 1996). The CO1 amplicons obtained from three (RC1-RC3) specimens were deposited in the
236
Genbank. In the present study, the phylogram constructed by the neighbour-joining method using
237
the COI barcoding gene suggested a clear separation between Ryphila cancellus and the other
238
closely related genera. Apart from their similar morphological characteristics with the closely
239
related genera, they are completely outgrouped in the phylogenetic tree. From the distance matrix
240
data, genetic distance between the selected individuals were clearly evident. Even though Phylira
241
and Ryphila shared almost similar morphological features, they exhibited greater genetic divergence
242
and exhibited a genetic distance of 0.7645.
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Different species assigned to various genera of Leucosiidae are known to inhabit sub-tidal and the
244
shelf regions of the Indo-Australian archipelago. Several related species are described from this area
245
and hence it would be worthwhile to conduct DNA barcoding in these species as well. In this
246
respect, the presence of the specimen belonging to Ryphila cancellus in the intertidal area is
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significant. Since this species was wrongly assigned to various other genera by previous authors,
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the COI barcoding would solve the taxonomic ambiguity of this genus.
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Acknowledgement
250
The authors would like to thank the Head, Department of Marine Biology, Microbiology and
251
Biochemistry and Nansen Environmental Research Centre (India) for the financial support and
252
facilities provided. Sequencing facility provided by Scigenom labs, Kerala, India is also greatly
253
acknowledged.
254
Conflict of interest
255
The authors declare that there are no potential conflicts of interest.
256
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Figure captions
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Table 1: The habitat and abundance of Ryphila cancellus (Herbst, 1783).
380
Figure 1: Ryphila cancellus, (Herbst, 1783) -Dorsal View (Male).
381
Figure 2: Ryphila cancellus, (Herbst, 1783)- Ventral view of male (a) and female (b).
382
Figure 3: Phylogenetic tree exhibiting the evolutionary relationship of Ryphila cancellus (Herbst,
383
1783) with other related genera.
384
Figure 4: Distance matrix tree displaying inter-generic divergence of Ryphila cancellus (Herbst,
385
1783) with closely related genera. The number of base substitutions per site between sequences
386
are shown. Analyses were conducted using the Kimura 2-parameter model.
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Table 1 Location
Latitude and longitude
Beach
Zone
Type of substratum
Payyambalam
11˚86’96’’N, 75˚35’21’’E
SandyIntermediate
Intertidal (lower littoral)
Coarse and loose sand
SandyDissipative
Intertidal (Mid-littoral and lower littoral ) Intertidal (Mid-littoral)
Fine and soft sediment
1
Specimens (number of specimens collected) 1
2
3
Muzhupilangadu 11˚79’63’’N, 75˚44’19’’E
3
1
Varkala
8˚73’42’’N, 76˚70’79’’E
4
2
Kovalam
8˚42’89’’N, 76˚95’81’’E
396
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397 398
402 403 404 405 406
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399 400
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Sl. No
SandyIntermediate
Sandy and rocky patchesIntermediate
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395
Intertidal (Mid-littoral)
Coarse (medium) sand Coarse (medium)
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Figure 1
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Figure 2
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Figure 3
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Figure 4
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Highlights
429
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The paper reports the DNA barcoding of crab Ryphila cancellus (Herbst, 1783)
431
Conducted taxonomic and molecular identification of the crab specimens
432
The CO1 gene of the crab was sequenced
433
The neighbor joining tree and distance matrix were plotted
434
Sequences were deposited in the Genbank as first molecular data of the species
435 436
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Conflict of interest declaration
439
pro of
440 441
The authors declare there are no potential conflict of interest in the following
443
manuscript titled “First barcode of Ryphila cancellus (Herbst, 1783), from the
444
southwest coast of India”
445
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