http://informahealthcare.com/mdn ISSN: 1940-1736 (print), 1940-1744 (electronic) Mitochondrial DNA, Early Online: 1–9 ! 2015 Informa UK Ltd. DOI: 10.3109/19401736.2015.1041114

RESEARCH ARTICLE

A molecular approach towards the taxonomy of fresh water prawns Macrobrachium striatum and M. equidens (Decapoda, Palaemonidae) using mitochondrial markers Deepak Jose1, B. Nidhin1, K. P. Anil Kumar1, P. J. Pradeep2, and M. Harikrishnan1

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CUSAT, School of Industrial Fisheries, Fine Arts Avenue, Kochi, Kerala, India and 2Department of Anatomy and Centex Shrimp, Faculty of Science, Mahidol University, Bangkok, Thailand Abstract

Keywords

Genus Macrobrachium includes freshwater prawns which inhabit most diverse habitats ranging from low saline areas to inland hill streams and impounded water bodies. Being morphologically conserved, this genus has been exposed to severe disputes related to their taxonomy, systematics and phylogeny. Macrobrachium striatum and M. equidens represent two morphologically related congeneric species within this genus. Earlier, M. striatum was considered as a striped form of M. equidens. Though these species are now well-described morphologically and differentiated into two species, no molecular level investigation has been carried out in support of their speciation. We report a study on M. striatum and M. equidens with emphasis to their molecular data through mitochondrial markers (16S ribosomal RNA and cytochrome oxidase subunit I). Results obtained from developed molecular markers of the two species revealed considerable genetic differentiation between them. Phylogram generated using Minimum evolution and Neighbour joining analyses differentiated M. striatum and M. equidens as two independent species. Genetic distance data showed high interspecific divergence (ranging from 3.9% to 17.0% for 16S rRNA sequences and 13.8% to 21.0% for COI sequences) between M. striatum and M. equidens confirming the findings of phylogram. Hence, it could be delineated that M. striatum and M. equidens represent two distinct species within genus Macrobrachium with emphasis to their morphology and genetics.

16S ribosomal RNA, cytochrome oxidase subunit I, Macrobrachium equidens, Macrobrachium striatum, taxonomy

Introduction Family Palaemonidae Rafinesque (1815) constitutes about 876 species within 116 genera forming one of the most unsolved groups of decapods in its systematics. Obstinate nature of this family in terms of its classification may be due to its ambient systematic history, convergence and divergence among and within species, taxonomic ambiguity and lack of proofread morphological database (Pileggi & Mantelatto, 2010). A typical example is the genus Macrobrachium Bate (1868) (Crustacea: Palaemonidae) with more than 240 species distributed worldwide (De Grave et al., 2008; Wowor et al., 2009). It includes freshwater prawns which inhabit most diverse habitats ranging from low saline areas to inland hill streams and impounded water bodies (Cook et al., 2002; Holthuis, 1952; Jalihal et al., 1993). They are thought to have descended from marine ancestors and subsequently occupied estuarine and freshwater areas. Presently this genus comprises two ecologically distinct groups – euryhaline and land locked. Euryhaline species complete their larval development in saline conditions while landlocked species complete their life cycle in freshwater (Munasinghe, 2010). Correspondence: Deepak Jose, CUSAT, School of Industrial Fisheries, Fine Arts Avenue, Fore Shore Road, Kochi 682016, Kerala, India. E-mail: [email protected]

History Received 18 January 2015 Revised 8 April 2015 Accepted 12 April 2015 Published online 29 June 2015

Morphological characters have largely been used for identification of Palaemonids till recently (Bray, 1976; Chace & Bruce 1993; Choy, 1984; Costa, 1979; Fincham, 1987; Holthuis, 1950; Holthuis, 1952; Jayachandran, 2001; Johnson, 1973; Riek, 1951) which, however, were difficult to characterize and required expertise of trained taxonomists. Being morphologically conserved, genus Macrobrachium confronted with severe disputes in their taxonomy, systematics and phylogeny. Rostrum and 2nd pereiopod were considered as the major characteristic features for establishing species groups. But these morphological characters acquired little phylogenetic value as they failed in ascertaining the monophyly of this genus (Liu et al., 2007). Even though rostrum and second pereiopod remained variable among species, this genus remained as one of the most taxonomically challenging decapods crustaceans (Munasinghe, 2010). Further, many of these traits are known to be influenced by environment and are not indicative of the underlying genetic divergence (Dimmock et al., 2004). The phylogenetic relationship among different species within the genus Macrobrachium remains vague. The greatest species diversity of Macrobrachium is reported from Indo-Pacific region (Murphy & Austin, 2005) and around 60 species are known from Indian subcontinent (Jayachandran & Indira, 2010; Pillai et al., 2014). It is also believed that there are numerous cryptic species in the genus (Cai et al., 2004; Cai & Ng 2002; Chace & Bruce, 1993; Short, 2004; Wowor & Choy, 2001).

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Six species of Macrobrachium inhabit Vembanad Lake, the largest estuarine system in the south-west coast of India viz. M. rosenbergii, M. idella, M. equidens, M. scabriculum and M. striatum (previously reported as M. striatus) (Suresh Kumar, 1998). Among these, M. striatum was considered as striped form of M. equidens (Jagadisha, 1977). Pillai (1990) differentiated M. striatum and M. equidens based on morphological characters, elevating M. striatum to an independent species. Later, Suresh Kumar (1998) investigated the systematics and bionomics of M. striatum. Though Jagadisha (1977) reported the occurrence

of M. equidens with longitudinal stripes along the entire body from Karwar region, this species is thought to be endemic to Kerala waters including Vembanad lake. No investigation has so far been conducted on M. striatum after Suresh Kumar (1998). Even though this species is well described morphologically, being a member of taxonomically confusing group, the necessity for molecular level investigation in support of its current description is inevitable considering the ‘‘error cascades’’ occurring due to bad taxonomic studies as reportd by Luiz et al. (2011). Recently, Murphy & Austin (2002, 2003, 2004, 2005) conducted molecular studies on Macrobrachium species with mitochondrial 16S ribosomal RNA gene marker. Furthermore, COI gene region was promoted as the best marker for a DNA bar coding system since it renders fast and accurate taxonomic identification (Hebert et al., 2003). Though molecular taxonomic studies were conducted on a number of Macrobrachium species, such studies on M. striatum were not attempted. We carried out an investigation on M. striatum and M. equidens with emphasis to their molecular data in support of morphological description through mitochondrial markers (16S ribosomal RNA and cytochrome oxidase subunit I).

Materials and methods Specimen collection About 9 different localities (Figure 1) were selected for collecting M. striatum and M. equidens during the period of July to October 2014 (Table 1). Previous morphological descriptions provided by Pillai (1990) helped to distinguish these animals morphologically at field itself (Figure 2). Specimens were transported to the laboratory and preserved at 20oC for obtaining 16S rRNA and COI sequences. Suitable candidates for molecular analysis were selected from morphologically congruent specimens of M. striatum and M. equidens obtained from selected sampling stations in a random fashion. Thirteen specimens of M. striatum (n ¼ 51) and 16 specimens of M. equidens (n ¼ 57) were selected for molecular analysis.

Figure 1. Map showing sampling areas.

Table 1. Details of sampling, mitochondrial markers developed and submitted in NCBI.

Taxon Macrobrachium striatum n ¼ 51 Total 16S rRNA sequences developed ¼ 13 Total COI sequences developed¼13

Macrobrachium equidens n ¼ 57 Total 16S sequences developed ¼ 16 Total COI sequences developed¼16

Sampling stations (Number of specimens collected (n) – Number of 16S sequence(s) developed – Number of COI sequence(s) developed )

16S (Accession number(s))

COI (Accession number(s))

Station Station Station Station Station Station Station Station Station Station Station Station Station Station Station Station Station Station

KM610144 – KM610146 KM610142-43 KM610139-41 KM610135-38 KM610145 KM610147 – KM593116 KM455124 KM593118-19 KM593115 KM593106-08 KM593110-14 KM593117 KM593109 KM593120

KM236798 – KM236801 KM236796-97 KM236793-95 KM236789-92 KM236800 KM236802 – KM255678 KM255667 KM255680-81 KM255677 KM255668-70 KM255672-76 KM255679 KM255671 KM255682

1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9

(3-1-1) (0-0-0) (2-1-1) (7-2-2) (14-3-3) (21-4-4) (3-1-1) (1-1-1) (0-0-0) (4-1-1) (1-1-1) (6-2-2) (2-1-1) (19-3-3) (22-5-5) (1-1-1) (1-1-1) (1-1-1)

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Genetic analysis Candidates for molecular analysis were subjected to DNA extraction using the DNeasy Blood and Tissue Kit (Qiagen) following the spin column protocol for purification of Total DNA from Animal Tissues. For amplifying partial 16S rRNA mitochondrial gene, primer pair (1471B-50 CCTGTTTANCAAAAACATGTCTG30 and 1472B-50 AGATAGAAACCAACCTGGCTCAC 30 ) and thermal profile were selected according to Liu et al. (2007). Thermal profile was slightly modified from the former. It consisted of an initial denaturation step for 5 min, at 94  C which was followed by 35 repeats of 94  C for 1 min, a gradient program of 45–55  C as annealing temperature, with an extension period of 1 min at 72  C and a final extension of 72  C for 5 min. For obtaining partial mitochondrial COI gene sequences, COI-f (50 -CCTGCAG GAGGAGGAGACCC-30 ) and COI-fR (50 -CGTCGTGGTAT GCCDTTTARWCCTA-30 ) (Liu et al., 2007) was used as primer pair. The temperature profile consisted of an initial step of 94  C for 5 min followed by 35 cycles of denaturation at 94  C for 1 min, annealing at 48–55  C for 1 min, extension at 72  C for 1 min and a final extension of 72  C for 5 min (Liu et al., 2007). Both genes (16S rRNA and mtCOI) were amplified using Sigma Aldrich ReadyMixÔ Taq PCR Reaction Mix (St. Louis, MO) with MgCl2 in Corbett gradient thermal cycler. Amplicons exhibiting intense bands after Agarose gel electrophoresis (1.2%) were selected for purification and sequencing. Sequence analysis Sequences were compiled using BioEdit 7.0.9 (Hall, 1999). Alignment was performed using Clustal X (Thompson et al., 1997). Different population genetic parameters were characterized using DnaSP 5.10 (Librado & Rozas, 2009) and Arlequin 3.1 (Excoffier, 2005). Phylogram (Minimum evolution (ME) and neighbour-joining (NJ) tree using 1000 bootstraps) and pair wise sequence distance between haplotypes were calculated using Kimura 2-Parameter model by MEGA 5 (Tamura et al., 2011).

Results A total of fifty eight sequences (29 16S rRNA and 29 COI) were developed successfully from samples collected from different

Figure 2. Macrobrachium striatum (a) lateral view; (b) dorsal view. Macrobrachium equidens (c) lateral view; (d) dorsal view.

DNA barcoding of Macrobrachium striatum and M. equidens

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Table 2. Details of sequences acquired from NCBI.

Taxon Macrobrachium equidens

Macrobrachium acanthurus

Alpheus angulosus

16S rRNA (Accession number(s))

COI (Accession number(s))

DQ194916 DQ194917 DQ194918 – – – GU929449 HM352445 HM352444 AY377838 – AF501637

AB235248 AB235249 AB235250 JF737756 JF737757 JF737758 JF737751 JF737752 JF737753 JF737754 JF737755 AF501655

Table 3. Details of genetic parameters analyzed for 16S rRNA sequences of Macrobrachium striatum and M. equidens.

Taxon Number of Sequences Alignment length Number of monomorphic sites Number of polymorphic sites Number of haplotypes Haplotype diversity (Hd) Nucleotide diversity (p) Mean number of pairwise difference Base frequency A C G T

Macrobrachium striatum

Macrobrachium equidens

13 531 base pairs (bp) 519

16 533 base pairs (bp) 525

12

8

7 0.731 ± 0.133 0.00396 ± 0.00265 2.10 ± 1.25

5 0.450 ± 0.151 0.00208 ± 0.00161 1.11 ± 0.76

34.59% 24.02% 12.97% 28.42%

29.67% 12.38% 23.09% 34.86%

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Figure 3. ME and NJ analyses based on 16S rRNA sequences showing relationship between Macrobrachium striatum, M. equidens and M. acanthurus. Numerical values represent corresponding bootstrap support (470) obtained after 1000 pseudo replications. Table 4. Genetic distance data obtained from 16S rRNA sequences.

Sl No 1 2 3 4 5

Taxon

Genetic distance (pairwise distance )

Overall mean distance

Within M. striatum Within M. equidens Between M. striatum and M. equidens Between M. striatum and M. acanthurus Between M. equidens and M. acanthurus

0.002–0.017 0.002–0.164 0.039–0.170 0.144–0.165 0.127–0.176

0.004 0.020 0.032 0.061 0.059

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stations using the mentioned primer pairs and protocols. Details of sequences developed for the study and submitted in NCBI are given in Table 1. All these sequences were devoid of insertions and deletions. Additional 16S rRNA and COI sequences of M. equidens were obtained from NCBI for analyzing the genetic congruence. Since Maciel et al. (2011) reported morphological proximity between. M. equidens and M. acanthurus, we also incorporated 16S rRNA and COI sequences of M. acanthurus in our analysis. Details of sequences acquired from NCBI are given in Table 2.

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16S rRNA sequence analysis For M. striatum, 13 16S rRNA sequences and for M. equidens, 16 16S rRNA sequences were developed. Final alignment of these sequences resulted sequences with 531base pairs for M. striatum and 533 base pairs for M. equidens. Considering M. striatum, 531base pairs consisted of 519 monomorphic and 12 polymorphic sites which constituted for 7 haplotypes within the species. In case of M. equidens sequences, 533 base pairs comprised of 525 monomorphic and 8 polymorphic sites, which accounted for 5 haplotypes within the species. Additional genetic parameters analyzed for both species are detailed in Table 3. In addition, 272 sites were parsimony informative. In order to analyze the relationship between M. striatum, M. equidens and M. acanthurus, phylogram (ME and NJ analyses) (Figure 3) was generated using Kimura 2 parameter method with 1000 pseudo replications. The cladistic array of corresponding 16S rRNA sequences in Minimum evolution and Neighbour joining trees supported the morphological identification. Primarily, bootstrap supports having lesser value than 70 were removed from phylogram. The genetic congruence of M. striatum was witnessed in phylogram as all of its 16S rRNA sequences exhibited an independent assemblage (possessing 100% bootstrap support) with respect to others. Macrobrachium equidens sequences developed for the present study (Table 1) and M. equidens sequences obtained from NCBI (Table 2) with accession numbers DQ194918 and DQ194916 were grouped together to form a monophyletic clade with 98% bootstrap value. Unexpected array was exhibited by M. equidens sequences with Accession no: DQ194917. M. acanthurus sequences also formed a single clade with 100% bootstrap support. The levels of intraspecific and interspecific divergence persisting within the analyzed sequences are detailed in Table 4. Sequence distance data reflected the findings from phylogram. Primarily, the 16S rRNA sequences of M. striatum showed intraspecific divergence ranging from 0.20% to 1.70% which remained under the threshold value of 3% for speciation, supporting the independent array of the same in ME and NJ analyses. In the case of M. equidens, intraspecific divergence ranged from 0.20% to 16.4% which was unexpected. On the contrary, the maximum intraspecific divergence in the sequences of M. equidens developed in the present study was 1.20% (not shown). Interspecific divergence between M. striatum and M. equidens was high, ranging from 3.9% to 17.0% which justified their diverged cladistic array in phylogram as two different taxa. Iinterspecific divergence for M. striatum and M. acanthurus ranged from 14.4% to 16.5%, which was high enough to differentiate them as individual species. Interspecific divergence between M. equidens and M. acanthurus (12.7–17.6%) was also high. As expected, Alpheus angulosus, the selected outgroup exhibited maximum genetic distance. COI sequence analysis A total of thirteen COI sequences for M. striatum and sixteen sequences for M. equidens were developed for analysis. Details of

DNA barcoding of Macrobrachium striatum and M. equidens

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Table 5. Details of genetic parameters analyzed for COI sequences of Macrobrachium striatum and M. equidens.

Taxon Number of Sequences Alignment length Number of monomorphic sites Number of polymorphic sites Number of haplotypes Haplotype diversity (Hd) Nucleotide diversity (p) Mean number of pairwise difference Base frequency A C G T

Macrobrachium striatum

Macrobrachium equidens

13 585 base pairs (bp) 574

16 603 base pairs (bp) 591

11

12

9 0.872 +/ 0.091 0.00443 +/ 0.0028 2.59 +/ 1.48

13 0.975 +/ 0.029 0.00482 +/ 0.0029 2.91 +/ 1.61

28.94% 24.73% 16.28% 30.05%

26.98% 23.82% 16.84% 32.36%

base pair length for each species are mentioned in Table 4. COI sequences for M. striatum constituted 585 base pairs (574 monomorphic and 11 polymorphic sites) accounting for 9 haplotypes within the species. In contrast, COI of M. equidens sequences were longer (603 base pairs with 591 monomorphic and 12 polymorphic sites) with 13 haplotypic representations and 78 parsimony informative sites. The subsequent genetic parameters of both species are given in Table 5. ME and NJ (Figure 4) analyses were performed for COI sequences using the same methodology done for 16S rRNA analysis. Results obtained from previous marker (16S rRNA) were very well reflected by COI gene sequences. Irrespective of an acquired COI sequence of M. equidens (Accession no: AB235249), rest of the COI sequences of M. striatum, M. equidens and M. acanthurus along with the selected outgroup A. angulosus exhibited independent assemblage with respect to their speciation. Overall bootstrap supports for nodes (Figure 4) were high as in previous phylogram using 16S rRNA (Figure 3). To confirm these results of COI sequences, Intraspecific and interspecific divergence were analyzed and results are given in Table 6. In distance data, the level of intraspecific divergence persisting within the sequences of M. striatum was limited (0.20– 1.20%) which supported the genetic identity of the same while interspecific distance between M. striatum and M. equidens accounted from 13.8% to 21.0%, which satisfied the results obtained from 16S rRNA regarding their corresponding speciation. As mentioned earlier, unexpected result was drawn out from COI distance table as the intraspecific divergence of M. equidens reached up to 22.9%. Maximum intraspecific divergence within our developed sequences of M. equidens was 1.0% (not shown). Range of genetic divergence encountered between M. striatum and M. acanthurus, M. equidens and M. acanthurus was very high (Table 6).

Discussion According to Gray et al. (2006), overall levels of lineages among taxa are addressed with incorporation of morphological, behavioral and molecular divergences. Since crustaceans are influenced by differential environmental conditions, there exists possibilty for phenotypic variations. Recently, Batista et al. (2014) investigated the population connectivity of Artemesia longinaris using COI gene. Across its distributional zone, the environmental conditions were diverse which resulted in variations of phenotypes, morphometric relations and reproductive periods. With the

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Figure 4. ME and NJ analyses based on COI sequences showing relationship between Macrobrachium striatum, M. equidens and M. acanthurus. Numerical values represent corresponding bootstrap support (470) obtained after 1000 pseudo replications.

assistance of marker gene (COI), it was found that there existed little genetic divergence within the species, except the reported variations that resolved the possibility of overestimation for speciation. Balasubramanian et al. (2014) revalidated the taxonomy of two species of genus Scylla viz. S. tranquebarica and S. serrata (seen as dark green and greenish brown colour morphs) from Indian waters through molecular studies that revealed

misidentifications and reciprocal monophyly persisting within them and inferred that S. tranquebarica reported from India was S. serrata. Macrobrachium striatum was thought to be a morphological variant (striped form) of M. equidens (Jagdisha, 1977) and was later established as an independant species based on morphology, breeding and larval development (Pillai, 1990). Morphologically,

DNA barcoding of Macrobrachium striatum and M. equidens

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Table 6. Genetic distance data obtained from COI sequences. Sl No

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1 2 3 4 5

Taxon

Genetic distance (pairwise distance )

Overall mean distance

Within M. striatum Within M. equidens Between M. striatum and M. equidens Between M. striatum and M. acanthurus Between M. equidens and M. acanthurus

0.002–0.012 0.002–0.229 0.138–0.210 0.186–0.196 0.192–0.215

0.004 0.027 0.080 0.082 0.081

this species is distinguished from M. equidens with the presence of green grayish brown longitudinal stripes along the entire length of body and absence of dark brown or greenish yellow mottling on carpus and palm. Even though morphological characters have largely been used for identification of Palaemonids (Bray, 1976; Chace & Bruce, 1993; Choy, 1984; Fincham, 1987; Holthuis, 1950, 1952; Riek, 1951), many of these traits are known to be influenced by environment and are not indicative of the underlying genetic divergence (Dimmock et al., 2004). In addition, conventional morphological approach fails to differentiate cryptic species complexes. Hence, genetic divergences have to be accounted for species identification (Pileggi et al., 2014). Examination of genetic, morphological and biogeographic characters will reveal the validity of obtained lineages as reproductively isolated population, species etc., on the basis of biological species concept (BSC). In addition, large-scale screening will provide critical first hand information on the presence of cryptic species which may be useful for generating systematic hypotheses in groups with incomplete taxonomies. Accounting all these limitations of conventional taxonomy based on morphology, DNA-based identification with the assistance of molecular markers have been utilized for resolving problems concerned with species identification, systematics, phylogenetic studies, determination of population structure, quantification of genetic variability etc (Lakra, 2013). Hebert et al. (2003) proposed mitochondrial markers as a remedy for resolving taxonomic ambiguity, confirming new speciation, forensic identifications, etc. Mitochondrial markers specifically 16S rRNA and COI are widely used in resolving the ambiguities in crustaceans (Balasubramanian et al., 2014). Phylogenic relationships of various species of Macrobrachium were extensively studied using different markers (Chen et al., 2009; Murphy & Austin, 2002, 2003, 2004, 2005; Murphy et al., 2004; Liu et al., 2007; Pereira et al., 1996). In the present study, mitochondrial markers (16S rRNA and COI) sequences were developed for M. striatum and M. equidens with a view to bring out the molecular variance between them. Barcoding of M. striatum has been attempted for the first time and the sequences developed for the present study (Table 1) constituted a single genetic entity. Within the 13 16S rRNA sequences developed for M. striatum, 12 polymorhic sites were determined which accounted for 7 haplotypes with haplotype diversity higher than diversity within nucleotides. COI data also reflected the same pattern. Avise (2004) and Grant et al. (2006) suggested that genetic diversity is influenced by many factors, including historical factors, anthropogenic activity, habitation and a low rate of mitochondrial evolution. Genetic diversity within a population can be well estimated from nucleotide diversity (Nei & Li, 1979). According to Grant & Bowen (1998), high haplotype diversity and low nucleotide diversity are indicative of population which had undergone rapid expansion followed by low effective population size. High-sequence divergence could be encountered between M. striatum and M. equidens as revealed from genetic distance table was accountable for providing a genetic level confirmation

for the existence of two distinct species. However, the divergence within the haplotypes of M. equidens was unanticipated, i.e. from 0.20% to 16.4% for 16S rRNA sequences and 0.20% to 22.9% in COI sequences. On close examination, it was found that 16S rRNA and COI sequences of M. equidens (Accession no. DQ194917 and Accession no. AB235249, respectively) showed higher degree of intraspecific divergence (i.e. 16.4% for 16S rRNA and 22.9% for COI) with respect to other haplotypes. These sequences were the derivatives of M. equidens from Singapore and could be attributed to a cryptic species as reported by Liu et al. (2007). This result corroborated with Goetze (2010) who stated that ‘‘examination of highly divergent mitochondrial lineages will provide some essential clue for the presence of cryptic species.’’ Luiz et al. (2011b) reported the ‘‘error cascades’’ occurring due to bad taxonomy in science. But recent technology like DNA barcoding offers accurate species identification and taxonomic assignment of morphologically identified individuals. Macrobrachium striatum and M. equidens were classified as two different species based on traditional taxonomy only. Results obtained from molecular markers in the present study showed considerable genetic differentiation between them which supported their present taxonomic classification. Hence, it could be delineated that M. striatum and M. equidens represents two distinct species within genus Macrobrachium with emphasis to their morphology and genetics.

Conclusion Earlier, traditional morphology was the only method for identification and differentiation of organisms. Traditional taxonomists were successful in identifying species to a great extent, but failed to establish the underlying genetic divegence as in cryptic speciation. Since modern techniques based on molecular markers offers an accurate method for species confirmation, implementing the same could be helpful in resolving the ambiguities regarding speciation. Our study recommends mitochondrial markers as efficient tool for revalidating the taxonomy of genus Macrobrachium.

Acknowledgements We gratefully acknowledge the Director, School of Industrial Fisheries, Cochin University of Science and Technology for providing necessary facilities and support for conducting this research.

Declaration of interest The authors declare responsibility for the entire contents of this paper and have no conflicts of interest.

References Avise JC. (2004). Molecular markers, natural history and evolution. 2nd edition. Sunderland, MA: Sinauer Associates. Balasubramanian CP, Cubelio SS, Mohanlal DL, Ponniah AG, Raj Kumar, Bineesh KK, Ravichandran P, et al. (2014). DNA sequence

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DOI: 10.3109/19401736.2015.1041114

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DNA barcoding of Macrobrachium striatum and M. equidens

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A molecular approach towards the taxonomy of fresh water prawns Macrobrachium striatum and M. equidens (Decapoda, Palaemonidae) using mitochondrial markers.

Genus Macrobrachium includes freshwater prawns which inhabit most diverse habitats ranging from low saline areas to inland hill streams and impounded ...
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