and morphological classification of Macrobrachium ...

See discussions, stats, and author profiles for this publication at: http://www.researchgate.net/publication/275174785

Mismatch between molecular (mtDNA) and morphological classification of Macrobrachium prawns from Southern Nigeria: Cryptic freshwater species and brackish water morphotypes ARTICLE in AQUACULTURE · OCTOBER 2013 Impact Factor: 1.83 · DOI: 10.1016/j.aquaculture.2013.06.013

DOWNLOAD

VIEWS

1

22

7 AUTHORS, INCLUDING: George F Turner

Cock Van Oosterhout

Bangor University

University of East Anglia

106 PUBLICATIONS 4,065 CITATIONS

72 PUBLICATIONS 5,942 CITATIONS

SEE PROFILE

SEE PROFILE

Bernd Hänfling University of Hull 38 PUBLICATIONS 1,180 CITATIONS SEE PROFILE

Available from: Bernd Hänfling Retrieved on: 07 August 2015

This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/authorsrights

Author's personal copy Aquaculture 410–411 (2013) 25–31

Contents lists available at SciVerse ScienceDirect

Aquaculture journal homepage: www.elsevier.com/locate/aqua-online

Mismatch between molecular (mtDNA) and morphological classification of Macrobrachium prawns from Southern Nigeria: Cryptic freshwater species and brackish water morphotypes Abayomi A. Jimoh a,b, Martins A. Anetekhai b, Steve Cummings a,1, Olatunji T.F. Abanikanda c, George F. Turner a,2, Cock van Oosterhout a,3, Bernd Hänfling a,⁎ a b c

Department of Biological Sciences, University of Hull, Hull, United Kingdom Department of Fisheries, Lagos State University, Ojo, Nigeria Department of Zoology, Lagos State University, Ojo, Nigeria

a r t i c l e

i n f o

Article history: Received 24 July 2012 Received in revised form 12 June 2013 Accepted 15 June 2013 Available online 23 June 2013 Keywords: Freshwater prawns Macrobrachium Nigeria Mitochondrial DNA Introgression, cryptic species Anthropogenic translocation

a b s t r a c t With a wide distribution across brackish and freshwater habitats in West African coastal regions, the giant prawns, Macrobrachium vollenhovenii and Macrobrachium macrobrachion, are potential candidates for aquaculture in the region. Here, we present the first molecular investigation of the phylogeography and systematics of these prawns. Morphological analyses unambiguously classed individuals into two clusters corresponding with the recognized species. However, phylogenies based on 3 mitochondrial DNA regions (CO1, 16S rRNA,12S rRNA) consistently recovered two highly divergent clades. One clade comprised all individuals from two geographically distant upstream (freshwater) populations of M. vollenhovenii, the other all individuals from brackish water sites, comprised of both morphospecies. Within mtDNA clades, there was no apparent genetic differentiation between morphospecies or geographic location, which is most consistent with gene flow through human-mediated translocation. Our results indicate a cryptic Macrobrachium species which appears to be adapted to freshwater conditions and therefore highly suitable for freshwater aquaculture. Further investigations are required to determine whether the existence of two apparent morphospecies in brackish water results from intraspecific polymorphism, recent speciation or extensive hybridization. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Freshwater prawns are important for commercial fisheries and aquaculture, with an estimated annual global production of around 300,000 t in 2001 (New, 2003), reportedly all belonging to Macrobrachium, the largest genus in the family Palaemonidae (New, 2002), with about 200 species so far identified (Jayachandran, 2001). Most species require brackish water during the initial stages of their life-cycle, and so are found in water that is directly or indirectly connected to the sea (New, 2003). However, populations of a few species, including Macrobrachium nipponense (Kutty, 2005), Macrobrachium australiense (Cook et al., 2002) and Macrobrachium vollenhovenii (Anetekhai, 1986; Marioghae, 1990) are believed to complete their entire life cycle in freshwater. ⁎ Corresponding author. Tel.: +44 1482 465804. E-mail address: b.haenfl[email protected] (B. Hänfling). 1 Present address: Anglia DNA Services Ltd., Norwich Research Park, Colney Lane, Norwich NR4 7UH, UK. 2 Present address: School of Biological Sciences, Bangor University, Gwynedd LL57 2UW, Wales, United Kingdom. 3 Present address: School of Environmental Sciences, University of East Anglia, Norwich Research Park, Norwich 7TJ, United Kingdom. 0044-8486/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aquaculture.2013.06.013

Macrobrachium are distributed worldwide in tropical and subtropical regions (New and Singholka, 1985) and occur throughout West Africa (Etim and Sankare, 1998), with four species reported from Nigeria (Bello-Olusoji et al., 2004): M. vollenhovenii — the African river prawn, Macrobrachium macrobrachion — the brackish water prawn, Macrobrachium felicinum — the Niger river prawn and Macrobrachium dux — the Congo river prawn. Despite their common names, all species are known to occur in brackish waters. These species can be reliably distinguished on the basis of their morphologies (Marioghae, 1982, 1987, 1990; Meye and Arimoro, 2005; Powell, 1982). Only the larger M. vollenhovenii and M. macrobrachion are considered to be of economic importance. Although both M. vollenhovenii and M. macrobrachion are potentially suitable for aquaculture, nothing is known of the distribution of genetic variability within and among natural populations, or of the phylogenetic relationships among species. Such questions are of interest for aquaculture because genetically isolated populations might also differ in quantitative traits (e.g. growth rate, size of maturity), or they might be adapted to different environments. Both species occur in a number of drainage systems with limited potential for migration between them. Such geographical isolation is typical for freshwater organisms often leading to pronounced genetic

Author's personal copy 26

A.A. Jimoh et al. / Aquaculture 410–411 (2013) 25–31

population structure (Hänfling and Brandl, 1998; Liu et al., 2011; Sharma and Hughes, 2009; Ward et al., 1994). Furthermore, M. vollenhovenii occupies a wide range of habitats ranging from coastal brackish water to upstream riverine environments, providing the potential for local adaptations. Genetic studies using mitochondrial DNA (mtDNA) have proven useful in addressing such questions (Avise, 1998, 2000; Murphy and Austin, 2005) and have been employed in a number of studies of crustaceans (e.g., de Bruyn et al., 2004a,b; Mather and de Bruyn, 2003; Murphy and Austin, 2004a,b; Pileggi and Mantelatto, 2010; Trontelj et al., 2004). This study aims to investigate whether genetically distinct populations of M. vollenhovenii and M. macrobrachion exist in Nigeria in order to provide information for the selection of aquaculture stocks and insights into the evolution of the genus Macrobrachium. We employed mtDNA sequence analyses of the CO1 and 16 s and 12S rRNA regions and morphological analysis to examine relationships between a number of freshwater and brackish water populations from different river systems in southern Nigeria.

2. Materials and methods 2.1. Sample collection and transportation Using baited non-return valve traps, samples of M. vollenhovenii and M. macrobrachion were collected from five sites in the southern region of Nigeria (Fig. 1 and Table 1): Asejire Lake and Ebonyi River are freshwater habitats from different river catchments with no freshwater connections between them; the remaining three sites are brackish water habitats, with two being from the same catchment. M. vollenhovenii was collected from all sites, but M. macrobrachion was found only at the three brackish water sites (Table 1). Fresh samples were preserved, at the point of collection, in 98% ethanol, transported to the laboratory and then stored at −20 °C.

Table 1 Details of sampling localities and sample sizes. Sample site

Asejire Lake Badagry Creek River Cross River Ebonyi River Cross

Site code

A B C E I

River catchment

Oyo Lagos Cross River Ebonyi Akwa-Ibom

Ecology

Freshwater Brackish Brackish Freshwater Brackish

Lat.

7° 6° 4° 5° 5°

08′ 43′ 10′ 59′ 01′

MV

MM

10/20 09/21 10/20 02/28 11/19

– 11/19 10/20 – 12/18

2.2. Molecular methods Three regions of the mitochondrial genome were investigated. The 12S rRNA region has provided resolution of deep divergences as well as among closely-related taxa (Goebel et al., 1999). The 16S rRNA region has been described as one of the most conserved mtDNA sequences (Schubart et al., 2000), useful for elucidating phylogenetic relationships in decapod crustaceans, including a broad range of Macrobrachium species (Munasinghe et al., 2003; Murphy and Austin, 2004b; Murphy et al., 2004; Nguyen and Austin, 2005; Pileggi and Mantelatto, 2010). Cytochrome oxidase c subunit 1 (CO1) is a highly variable region, widely used for phylogeographic and phylogenetic studies of more recent events (Liu et al., 2011; Sharma and Hughes, 2009). In the first instance the 12S region of 5–8 individuals per population was sequenced. Subsequently, a subset of individuals representing the major lineages and all populations was sequenced for 16S and CO1 to attempt finer resolution, and to test whether the results were influenced by PCR artifacts or amplification of pseudogenes (Table 2). Tissue samples were obtained from either the distal segment of the 2nd pair of walking legs (pereiopods) or the tail muscle. Total DNA was extracted using the cetyl-trimethyl ammonium bromide (CTAB) extraction protocol (Doyle and Doyle, 1985). A fragment of

8°

Asejire Lake (A) Ebonyi River (E) River Cross, Itu (I)

0

100km

River Cross, Calabar (C) 4° 5°

4° 2° 8° 7° 8°

Sample Size (M/F)

M = Male; F = Female; MV = Macrobrachium vollenhovenii; MM = M. macrobrachion.

12°

Badagry Creek (B)

22′ 26′ 54′ 58′ 13′

Long.

10°

14°

Fig. 1. Map of Nigeria showing sampling sites. Populations are color-coded according to the legend in Fig. 3.

Author's personal copy A.A. Jimoh et al. / Aquaculture 410–411 (2013) 25–31

approximately 700 bp of CO1 mtDNA was initially amplified using the universal primers LCO1490 (5′-GGT CAA CAA ATC ATA AAG ATA TTG G-3′) and HCO2198 (5′-TAA ACT TCA GGG TGA CCA AAA AAT CA-3′) (Folmer et al., 1994) and sequenced according to the protocol below. Because of the unsatisfactory amplification of a number of samples, these sequences were used to design more specific primers LCO new b (5′-TCT CAA CAA ACC ATA AAG ACA TTG-3′) and H711 (5′-GTT AAA ATG TAT ACT TCT GGG TGC C-3′) which were used to amplify a 669 bp region of the remaining individuals. The 16S rRNA mtDNA was amplified using the primers 16SAR (5′-CGC CTG TTT ATC AAA AAC AT-3′) and 16SBR (5′-CCG GTC TGA ACT CAG ATC ACG T-3′) (Kessing et al., 1989) while the primers 12S LEV and 12S DES1 were used to amplify the 12S rRNA region. Polymerase chain reaction (PCR) for all the three regions was performed in a 20 μl reaction volume in a MJ Research Peltier Thermal Cycler. Each 20 μl reaction volume consisted of 2 μl of 10× NH4 buffer (Bioline), 0.6 μl 50 mM MgCl2 (Bioline), 2 μl of 2 mM dNTPs, 0.4 μl of each 10 mM primer dilution, 0.1 5U μl−1 Taq polymerase (Bioline), 10–50 ng of DNA template and 12.5 μl double-distilled water. The PCR profile consisted of an initial denaturation step (94 °C for 3 min.), 36 cycles of denaturation (94 °C for 45 s.), annealing (50 °C for 45 s.) and extension (72 °C for 1 min.), followed by a final extension (72 °C for 30 min.). CO1 mtDNA PCR products were cloned into pGEM®-T Easy vector (Promega) and transformed into Escherichia coli DH5α competent cells (Invitrogen). Transformed colonies were screened using the PCR protocol with initial denaturation step of 95 °C for 5 min, followed by 30 cycles of denaturation (94 °C for 1 min.), annealing (54 °C for 1 min.) and extension (72 °C for 1 min.), and a final extension at 72 °C for 5 min. The positive colonies were sequenced directly from the PCR products using the sequencing conditions: 30 cycles of 96 °C for 20 s, 50 °C for 20 s, and 60 °C for 4 min. The 16S and 12S regions were, however, sequenced directly from the respective PCR products using the same sequencing conditions as for the CO1 mtDNA. The fragments were separated on a Beckman Coulter CEQ™ 8000 Genetic Analysis System. 2.3. Genetic data analysis Sequence chromatograms were viewed and edited manually using the software CodonCode Aligner (version 1.5.2) (Codon Code Corporation, Dedham, Massachusetts, USA). The beginning and end of the CO1, 12S and 16S regions were confirmed by comparing with the published sequences of Macrobrachium rosenbergii. Once edited, multiple alignments were performed using ClustalW in MEGA Version 3.1 (Kumar et al., 2004). The most suitable model of sequence evolution for each region was obtained using ModelGenerator v 0.82 (Keane et al., 2006). Maximum-likelihood analyses were performed with PHYML v2.4.4 (Guindon and Gascuel, 2003). Parameters of the models of sequence evolution and rate heterogeneity were estimated by PHYML. Bootstrapping (1000 replicates, Felsenstein, 1985) was used to estimate support for the resulting topologies. Pairwise nucleotide distances within and between clades were generated using the Kimura-2-parameter (Kimura, 1980) model of substitution. We used Network v. 4.5.10 (Bandelt et al., 1999) to construct a median-joining haplotype network. All other Table 2 Sample size used for phylogenetic analysis. Sample Macrobrachium Macrobrachium Macrobrachium Macrobrachium Macrobrachium Macrobrachium Macrobrachium Macrobrachium

vollenhovenii, Asejire vollenhovenii, Badagry vollenhovenii, Calabar vollenhovenii, Ebonyi vollenhovenii, Itu macrobrachion, Badagry macrobrachion, Calabar macrobrachion, Itu

12S

16S

CO1

8 7 7 8 5 7 6 6

7 6 3 6 4 6 3 3

2 2 2 2 2 3 3 3

27

nucleotide positions were weighted at 50 and we used an ε value of 0. Furthermore, transversions were weighted three times higher than transitions to decrease the likelihood of homoplastic substitutions. 2.4. Morphological analysis Morphological differentiation among populations was investigated using six traits previously used for identification of Macrobrachium species (Botello and Alvarez, 2006; Dimmock et al., 2004; Enin, 1994; Jimoh et al., 2005; Mantelatto and Barbosa, 2005; Mariapann and Balasundaram, 2004; Meye and Arimoro, 2005). Three morphometric traits (chelal length, ChL; chelal width, ChW; rostrum length, RL) and two meristic traits (numbers of dorsal rostral spines, DS, and ventral rostral spines, VS) were scored for all individuals (Table 1). Carapace length (CpL) was used as a surrogate of body size. The analysis was carried out separately for males and females due to the pronounced sexual dimorphism in both species. For this analysis, the sample of male M. vollenhovenii from the Ebonyi River was excluded because of the small sample size. All morphometric variables were tested for the assumptions of normality and homogeneity of variance, using the Kolmogorov–Smirnov test for goodness-of-fit, and the Levene's test for homogeneity of variance (Sokal and Rohlf, 1995). All morphometric variables had to be log-transformed to satisfy the conditions for parametric testing. Chelal width was excluded from further analyses, as it did not prove possible to transform this variable to meet the assumptions of normal distribution and homoscedasticity. Covariance analysis showed that all remaining morphometric characters were significantly correlated with size (data not shown). The relationship between the rostral length and carapace length of females differed among populations, so that a common correction for size could not be carried out, and this character was also excluded from further analysis. A general linear model (GLM) was used to test for the effect of body size, in which sites were treated as fixed factors and carapace length as covariate. Variables which showed a significant effect of body size were corrected for size by calculating the residuals from the pooled within-site slope (Thorpe, 1976). Residuals and raw meristic characters were then subjected to univariate and multivariate analyses using the appropriate procedures in SPSS v 16.0. Differences among populations and taxa in distribution patterns of rostral spines were tested using χ2-tests. Parametric analysis of variance (ANOVA) was used to test for differences among populations and morphospecies in transformed morphometric data. Discriminant Function Analysis was performed entering all independent variables together and using populations as groups. 3. Results 3.1. Genetic diversity A 669 base-pair fragment of CO1 sequence was successfully amplified from 19 specimens of M. vollenhovenii and M. macrobrachion (Table 2). Eighteen unique haplotypes were identified (Haplotype diversity, H = 0.994), of which one was shared between M. vollenhovenii and M. macrobrachion. The haplotypes contained 179 variable sites with no insertions or deletions, suggesting the absence of pseudogenes. A 540 bp fragment of the 12S gene region, with 135 variable sites, was amplified for 54 specimens of M. vollenhovenii and M. macrobrachion. Of these, 31 unique haplotypes were recorded (H = 0.905). Two common haplotypes were shared between both species, while 23 haplotypes were unique to M. vollenhovenii (H = 0.934), and 11 were found only in M. macrobrachion (H = 0.830). For the 16S region, the sequence was 463 bp, with 76 of the sites being variable. Eighteen haplotypes were recorded from 38 individuals of M. vollenhovenii and M. macrobrachion (H = 0.836). Similar to the S12 region, M. vollenhovenii showed a higher haplotype diversity in the S16 region than M. macrobrachion (H = 0.926 and H = 0.455, respectively). The

Author's personal copy 28

A.A. Jimoh et al. / Aquaculture 410–411 (2013) 25–31

P b 0.001). The first and second discriminant functions capture 99% of the variance in the male dataset and 98% variance in the female dataset. Despite the algorithm being set to discriminate among populations, both males and females were clearly separated according to morphological species (Fig. 4), being classified correctly 100% of the time, but they were only weakly classified by population (70% for males and 50% for females).

Table 3 Summary of sequence characteristics of 3 mitochondrial gene regions. Gene region

12S

16S

CO1

Number of samples Length of sequences (bp) Base frequency: A C G T Transition/transversion Model of evolution I

54 540

38 463

19 669

0.29 0.11 0.22 0.38 4.09 TIM + I 0.64

0.35 0.25 0.12 0.28 2.40 GTR + I 0.70

0.29 0.28 0.17 0.26 6.15 TrN + I 0.69

4. Discussion We found that individuals could be clearly assigned on the basis of morphology to two distinct groups corresponding with the recognized morphological species M. vollenhovenii and M. macrobrachion (Botello and Alvarez, 2006; Dimmock et al., 2004; Enin, 1994; Jimoh et al., 2005; Mantelatto and Barbosa, 2005; Mariapann and Balasundaram, 2004; Meye and Arimoro, 2005). By contrast, molecular phylogenetic analyses revealed two clades which were not concordant with traditional taxonomy, but rather clustered together individuals from the same environment (i.e., freshwater vs. brackish water). Thus, the brackish water clade comprised individuals of both morphospecies, M. vollenhovenii and M. macrobrachion from Badagry Creek and the Calabar and Itu axes of the River Cross. For reproduction, Macrobrachium species usually move from freshwater to brackish water, but our study suggests that the freshwater populations of M. vollenhovenii may have been geographically isolated from the other populations in this study. The relatively high levels of mtDNA differentiation suggest that the freshwater populations might be considered to be a separate cryptic species. Such an upstream speciation from a marine ancestor appears to be commonplace in Asian Macrobrachium species (Wowor et al., 2009) but had not been reported for African freshwater prawns. Given a calibrated estimate of the rate of sequence evolution in a comparable taxon, the date since divergence can be estimated. Knowlton et al. (1993) and Shanks et al. (1995) reported a rate of sequence evolution for CO1 mtDNA gene in caridean crustaceans from opposite sides of the Isthmus of Panama to be between 2.2 and 2.6% divergence per million years. Applying these rates of sequence evolution would date the divergence between the freshwater and brackish water clades (using the mean sequence divergence of 19.9%) at between 7.6 and 9.1 Myr, well into the Miocene.

nucleotide substitution models estimated to be most appropriate are listed in Table 3, along with other relevant parameters. 3.2. Phylogeny reconstruction Phylogenetic analysis and network analysis of all three mtDNA regions produced trees with similar topology showing (i) a clear deep split between freshwater and brackish water populations; (ii) no differentiation between brackish water individuals assigned to the morphospecies M. vollenhovenii or M. macrobrachion; (iii) little geographical structure within either freshwater or brackish water clades (Figs. 2, 3). Mean (±SEM) percentage sequence divergence was more than an order of magnitude smaller within clades [0.700 (±0.173) %] than between clades [15.83 (±2.54) %]. However despite the lack of distinct clades within the two major lineages, only few haplotypes were shared among populations. There was no consistency between gene regions in whether the freshwater or brackish clades harbored the greater level of sequence divergence (Table 4). 3.3. Morphological analysis Univariate analysis (ANOVA) showed that in both datasets all meristic and morphometric characters showed highly significant differences among populations (ChL: F7,157 = 4.38, P b 0.001; RL: F7,157 = 92.83, P b 0.001; DRS: F7,157 = 167.25, P b 0.001; VRS: F7,157 = 33.49,

B C B I C I I C B C B I B C

100

100

I

Macrobrachium vollenovenii Macrobrachium macrobrachion A= Asejire Lake B= Badagry Creek I= Itu (River Cross) E= Ebonyi River C= Calabar (River Cross)

A A E E M. rosenbergii

0.02 Fig. 2. Maximum-likelihood (ML) tree for CO1 of Macrobrachium vollenhovenii, M. macrobrachion and M. rosenbergii (the outgroup). Bootstrap probabilities, in %, for 1000 replicates (ML) are indicated (N70%).

Author's personal copy A.A. Jimoh et al. / Aquaculture 410–411 (2013) 25–31

29

Fig. 3. Medium joining network of COI (a) 12S (b) and 16S (c) haplotypes from Macrobrachium macrobrachion (red color shades) and M. vollenhovenii from freshwater locations (green color shades) and brackish water locations (blue color shades). Adjacent haplotypes are connected through a single point mutation. Each circle represents a single haplotype and its diameter is proportional to the number of individuals with that haplotype. The color codes represent the locations in which the haplotype is found, black squares represents unsampled haplotypes.

Interestingly, the M. vollenhovenii and M. macrobrachion morphospecies from the brackish habitats could not be distinguished on the basis of mtDNA. This might indicate that these well-characterized morphological species are in fact conspecific morphs, or that they are the result of very recent ecological speciation, or that M. macrobrachion mtDNA has introgressed into M. vollenhovenii. In the absence of nuclear genetic

data it is not possible to distinguish among these three scenarios, yet we want to discuss the plausibility of these hypotheses. Given that male morphotypes are commonly observed in freshwater prawns (Rojas et al., 2012), including Macrobrachium (Moraes-Riodades and Valenti, 2004; Nagamine and Knight, 1980), the observed pattern could be parsimoniously explained by intra-specific variation. However,

Author's personal copy 30

A.A. Jimoh et al. / Aquaculture 410–411 (2013) 25–31

Table 4 Mean sequence divergences within and among clades for 3 mitochondrial regions. Region

Freshwater clade

Brackish clade

Between clades

CO1 12-S rRNA 16-S rRNA

1.1% 1.3% 0.2%

0.5% 0.4% 0.7%

19.9% 17.6% 10.8%

our results also show the existence of two distinct female morphotypes which, to the best of our knowledge, has not been reported in other freshwater prawns. The presence of polymorphism in both sexes suggests ecological drivers rather than different male mating strategies. Ecotypes have been reported in many freshwater fish including salmonids (Pigeon et al., 1997) and sticklebacks (McPhail, 1993), and in many cases, reproductive isolation between ecotypes has created sympatric species pairs that are indistinguishable based on mtDNA. A third scenario that could also explain the absences of differentiation at mtDNA between distinct morphotypes is complete mitochondrial introgression through hybridization after secondary contact. Although most Macrobrachium species do not hybridize by natural mating (Fu et al., 2004), Sankolli et al. (1982) reported a successful natural mating between M. rosenbergii and Macrobrachium malcolmsonii. Likewise, Shokitai (1978) obtained hybrids from natural mating between Macrobrachium asperulum and Macrobrachium shokitai. However, extensive and continuous hybridization over many generations would be required to lead to such a complete

a) males 4 3

DF 2 (4.3%)

2 1

introgression across several river systems. If M. vollenhovenii colonized the brackish water habitat, its initially small founder population size may have increased the rate of hybridization. The resulting genetic drift and/or selection for an mtDNA haplotype better adapted to brackish habitats might have promoted fixation of the M. macrobrachion haplotypes in individuals with a largely M. vollenhovenii genotype. Possibly, this could explain complete mitochondrial introgression through hybridization after secondary contact. The lack of geographic structuring among populations from habitats of the same salinity is consistent with a single origin of the freshwater populations through upstream speciation (Wowor et al., 2009). Given that freshwater organisms often show pronounced genetic differentiation among drainage systems (Hänfling and Brandl, 1998; Liu et al., 2011; Sharma and Hughes, 2009; Ward et al., 1994), it was expected that there would be geographic structuring between samples collected from Badagry Creek and Asejire Lake, on one hand, and Ebonyi, Itu and Calabar, on the other. After all, both areas are not only geographically distinct and lack freshwater connections, but they also lie on opposite sides of the Niger River system. The absence of such differentiation suggests recent connections between the river systems to the east and west of the Niger, or more plausibly, that gene flow though human-mediated transfer between habitats of similar salinity has occurred, which negates any geographic structuring. In Nigeria, provision of brackish water for prawn culture is very expensive and has been a factor inhibiting the development of prawn culture. The results from this study clearly show that there are distinct fully freshwater prawns (M. vollenhovenii) that attain a substantial adult size, which implies that wholly freshwater culture can be developed. Furthermore, we suggest that further studies using nuclear markers are needed to clarify the evolutionary relationship between the two freshwater morphospecies. A formal description of the cryptic freshwater species and a thorough re-evaluation of the taxonomic key which is used to distinguish these species are also necessary.

0 -1

Acknowledgments

-2 -3 -4 -8

-6

-4

-2

0

2

4

6

8

DF 1 (94.7%)

b) Females

This study was made possible with a Split-Site scholarship, awarded to Abayomi Jimoh, by the Commonwealth Scholarship Commission in the United Kingdom. The authors acknowledge the technical support offered by members of the Molecular Ecology and Fisheries Genetics Laboratory, University of Hull, United Kingdom. CvO was funded by the Earth and Life Systems Alliance (ELSA). We are also grateful to Mr. M.A. Ajibade who helped with the collection of samples, as well as Dr. H.A. Fashina-Bombata and Mr. R.G. Ajepe.

4 3

References

CDF 2 (3.4%)

2 1 0 -1 -2 -3 -4 -6

-4

-2

0

2

4

6

8

10

CDF 1 (94.9%) Fig. 4. Ordination of canonical variates of a Discriminant Function Analysis of four morphological characters. Individuals are marked as white symbols (M. macrobrachion) or black symbols (M. vollenhovenii). Within each species, individuals are marked with different symbols for each site. Large symbols represent group centroids.

Anetekhai, M.A., 1986. Aspects of the Bioecology of the African River Prawn, Macrobrachium vollenhovenii (Herklots), in Asejire Lake, Oyo State, Nigeria. University of Ibadan, Nigeria, p. 225 (PhD Thesis). Avise, J.C., 1998. The history and purview of phylogeography: a personal reflection. Molecular Ecology 7, 371–379. Avise, J.C., 2000. Phylogeography: The History and Formation of Species. Harvard University Press, Cambridge, Massachusetts. Bandelt, H.-J., Forster, P., Röhl, A., 1999. Median-joining networks for inferring intraspecific phylogenies. Molecular and Biological Evolution 16, 37–48. Bello-Olusoji, A.O., Ariyo, T.O., Arinola, A., 2004. Taxonomical studies on rocky freshwater prawns at Erin-Ijesha waterfalls. Journal of Food, Agriculture and Environment 2 (issues 3 and 4), 280–283. Botello, A., Alvarez, F., 2006. Allometric growth in Creaseria morleyi (Creaser, 1936) (Decapoda: Palaemonidae) from the Yutacan Peninsula, Mexico. Caribbean Journal of Science 42 (2), 171–179. Cook, B.D., Bunn, S.E., Hughes, J.M., 2002. Genetic structure and dispersal of Macrobrachium australiense (Decapoda: Palaemonidae) in western Queensland, Australia. Freshwater Biology 47, 2098–2112. de Bruyn, M., Wilson, J.A., Mather, P.B., 2004a. Huxley's line demarcates extensive genetic divergence between eastern and western forms of the giant freshwater prawn, Macrobrachium rosenbergii. Molecular Phylogenetics and Evolution 30, 251–257.

Author's personal copy A.A. Jimoh et al. / Aquaculture 410–411 (2013) 25–31 de Bruyn, M., Wilson, J.C., Mather, P.B., 2004b. Reconciling geography and genealogy: phylogeography of giant freshwater prawns from the Lake Carpentaria region. Molecular Ecology 13, 3515–3526. Dimmock, A., Williamson, I., Mather, P., 2004. The influence of environment on the morphology of Macrobrachium australiense (Decapoda: Palaemonidae). Aquaculture International 12, 435–456. Doyle, J.J., Doyle, J.L., 1985. A rapid DNA isolation procedure for small amounts of fresh leaf tissue. Phytochemical Bulletin 19, 11–15. Enin, U.I., 1994. Length–weight parameters and condition factor of two West African prawns. Review of Hydrobiology Tropics 27 (2), 121–127. Etim, L., Sankare, Y., 1998. Growth and mortality, recruitment and yield of the freshwater shrimp, Macrobrachium vollenhovenii, Herklots 1851 (Crustacea, Palaemonidae) in the Fahe reservoir, Cote d'Ivoire, West Africa. Fisheries Research 38, 211–223. Felsenstein, J., 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783–791. Folmer, O., Black, M., Hoeh, W., Lutz, R., Vrijenhoek, R., 1994. DNA primers for amplification of mitochondrial cytochrome C oxidase subunit 1 from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology 3 (5), 294–299. Fu, H., Gong, Y., Wu, Y., Xu, P., Wu, C., 2004. Artificial interspecific hybridization between Macrobrachium species. Aquaculture 232, 215–223. Goebel, A.M., Donnelly, J.M., Atz, M.E., 1999. PCR primers and amplification methods for 12S ribosomal DNA, the control region, cytochrome b in bufonids and other frogs, and an overview of PCR primers which have amplified DNA in amphibians successfully. Molecular Phylogenetics and Evolution 1, 163–199. Guindon, S., Gascuel, O., 2003. A simple, fast and accurate algorithm to estimate large phylogenies by maximum likelihood. Systematic Biology 52, 696–704. Hänfling, B., Brandl, R., 1998. Genetic differentiation of the bullhead Cottus gobio L. across watersheds in central Europe: evidence for two taxa. Heredity 80, 110–117. Jayachandran, K.V., 2001. Palaemonid Prawns: Biodiversity, Taxonomy, Biology and Management. Enfield Science Publishers. Jimoh, A.A., Fakoya, K.A., Hammed, A.M., Amosu, A.O., Kumolu-Johnson, C.A., 2005. Meristics and morphometrics in the African river prawn, Macrobrachium vollenhoveni (Herklots, 1857) from Ologe Lagoon, Southwest, Nigeria. Journal of Agricultural and Environmental Research Studies (1), 12–18. Keane, T.M., Creevey, C.J., Pentony, M.M., Naughton, T.J., McInerney, J.O., 2006. Assessment of methods for amino acid matrix selection and their use on empirical data shows that ad hoc assumptions are not justified. BMC Evolutionary Biology 6, 29. Kessing, B., Croon, H., Martin, A., McIntosh, C., Owen Mcmillan, W., Palumbi, S., 1989. The Simple Fool's Guide to PCR. University of Hawaii, Honolulu. Kimura, M., 1980. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. Journal of Molecular Evolution 16, 111–120. Knowlton, N., Weigt, L.A., Solorzan, L.A., Mills, D.K., Bermingham, E., 1993. Divergence in proteins, mitochondrial DNA, and reproductive compatibility across the Isthmus of Panama. Science 260, 1629–1632. Kumar, S., Tamura, K., Nei, M., 2004. MEGA 3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Briefings in Bioinformatics 5, 150–163. Kutty, M.N., 2005. Towards sustainable freshwater prawn aquaculture — lessons from shrimp farming, with special reference to India. Aquaculture Research 36, 255–263. Liu, M.Y., Tzeng, C.S., Lin, H.D., 2011. Phylogeography and the genetic structure of the land-locked freshwater prawn Macrobrachium asperulum (Crustacea: Decapoda: Palaemonidae) in Taiwan. Hydrobiologia 671, 1–12. Mantelatto, F.L.M., Barbosa, L.R., 2005. Population structure and relative growth of freshwater prawn Macrobrachium brasiliense (Decapoda: Palaemonidae) from Sao Paulo State, Brazil. Acta Limnology Brasileira 17 (3), 245–255. Mariapann, P., Balasundaram, C., 2004. Studies on the morphology of Macrobrachium nobili (Decapoda: Palaemonidae). Brazilian Archives of Biology and Technology 47 (3), 441–449. Marioghae, I.E., 1982. Notes on the biology and distribution of Macrobrachium vollenhovenii and Macrobrachium macrobrachion in the Lagos Lagoon. Reviews in Zoology of Africa 96 (3), 493–508. Marioghae, I.E., 1987. An appraisal of the cultivability of Nigerian palaemonid prawns. African Regional Aquaculture Centre (ARAC) Working Paper ARAC/87/WP/4, p. 11. Marioghae, I.E., 1990. Studies on fishing methods, gear and marketing of Macrobrachium in the Lagos Area. Nigerian Institute for Oceanography and Marine Research (NIOMR) Technical Paper No. 53, p. 20. Mather, P.B., de Bruyn, M., 2003. Genetic diversity in wild stocks of the giant freshwater prawn (Macrobrachium rosenbergii): implications for aquaculture and conservation. NAGA, WorldFish Centre Quarterly 26 (4), 4–20. McPhail, J.D., 1993. Ecology and evolution of sympatric sticklebacks (Gasterosteus): origin of the species pairs. Canadian Journal of Zoology 71, 515–523. Meye, J.A., Arimoro, F.O., 2005. Aspects of the ecology, reproductive and growth characteristics of Macrobrachium dux (Lenz, 1910) (Crustacea: Decapoda: Natantia) in Orogodo River, Niger Delta, Nigeria. European Journal of Scientific Research 2 (3), 585–596.

31

Moraes-Riodades, P.M.C., Valenti, W.C., 2004. Morphotypes in male Amazon river prawns, Macrobrachium amazonicum. Aquaculture 236, 297–307. Munasinghe, D.H.N., Murphy, N.P., Austin, C.M., 2003. Utility of mitochondrial DNA sequences from four gene regions for systematic studies of Australian freshwater crayfish of the genus Cherax (Decapoda: Parastacidae). Journal of Crustacean Biology 23, 402–417. Murphy, N.P., Austin, C.M., 2004a. Phylogeography of the widespread Australian freshwater prawn, Macrobrachium australiense (Decapoda, Palaemonidae). Journal of Biogeography 31, 1065–1072. Murphy, N.P., Austin, C.M., 2004b. Multiple origins of the endemic Australian Macrobrachium (Decapoda: Palaemonidae) based on 16S rRNA mitochondrial sequences. Australian Journal of Zoology 52 (5), 549–559. Murphy, N.P., Austin, C.M., 2005. Phylogenetic relationships of the globally distributed freshwater prawn genus Macrobrachium (Crustacea: Decapoda: Palaemonidae): biogeography, taxonomy and the convergent evolution of abbreviated larval development. Zoologica Scripta 34 (2), 187–197. Murphy, N.P., Short, J.W., Austin, C.M., 2004. Re-examination of the taxonomy of the Macrobrachium australiense Holthius (Decapoda: Palaemonidae) species-complex: molecular evidence for a single species. Invertebrate Systematics 18 (2), 227–232. Nagamine, C.M., Knight, A.W., 1980. Development, maturation, and function of some sexually dimorphic structures of the Malaysian prawn, Macrobrachium rosenbergii (De Man) (Decapoda, Palaemonidae). Crustaceana 39, 141–152. New, M.B., 2002. Farming freshwater prawns: a manual for the culture of the giant river prawn (Macrobrachium rosenbergii). FAO Fisheries Technical Paper 428, 212. New, M.B., 2003. The role of freshwater prawns in sustainable aquaculture. In: Purushan, K.S., Kumar, M.B., Dinesh, K. (Eds.), Freshwater Prawns 2003 — Souvenir of the International Symposium on Freshwater Prawns- 2003, Cochin, 20–23 August 2003. College of Fisheries, Kerala Agricultural University, Kochi, India, pp. 10–13. New, M.B., Singholka, S., 1985. Freshwater prawn farming: a manual for the culture of Macrobrachium rosenbergii. FAO Fisheries Technical Paper No. 225, p. 116. Nguyen, T.T.T., Austin, C.M., 2005. Phylogeny of the Australian freshwater crayfish Cherax destructor-complex (Decapoda: Parastacidae) inferred from four mitochondrial gene regions. Invertebrate Systematics 19, 209–216. Pigeon, D., Chouinard, A., Bernatchez, L., 1997. Multiple modes of speciation involved in the parallel evolution of sympatric morphotypes of lake whitefish (Coregonus clupeaformis, Salmonidae). Evolution 51, 196–205. Pileggi, L.G., Mantelatto, F.L., 2010. Molecular phylogeny of the freshwater prawn genus Macrobrachium (Decapoda, Palaemonidae), with emphasis on the relationships among selected American species. Invertebrate Systematics 24, 194–208. Powell, C.B., 1982. Fresh and brackish water shrimps of economic importance in Niger Delta. 2nd Annual Conference of the Fisheries Society of Nigeria, held at Calabar, 22–24 January, 1982. Rojas, R., Morales, M.C., Rivadeneira, M.M., Thiel, M., 2012. Male morphotypes in the Andean river shrimp Cryphiops caementarius (Decapoda: Caridea): morphology, coloration and injuries. Journal of Zoology 288, 21–32. Sankolli, K.N., Shenoy, S., Jalihal, D.R., Almelkar, G.B., 1982. Cross-breeding experiment with the giant freshwater prawn, Macrobrachium rosenbergii and M. malcolmsonii. In: New, M.B. (Ed.), Developments in Aquaculture and Fisheries Sciences 10: Giant Prawn Farming. Elsevier, Amsterdam, pp. 91–98. Schubart, C.D., Neigal, J.E., Felder, D.L., 2000. Use of the mitochondrial 16S rRNA gene for phylogenetic and population studies of Crustacea. In: Schram, F.R. (Ed.), Crustacean issues 12. The Biodiversity Crisis and Crustacea. A.A. Balkerna, Rotterdam. Shanks, T.M., Black, M.B., Halanych, K.M., Lutz, R.A., Vrijenhoek, R.C., 1995. Miocene radiation of deep-sea hypothermal vent shrimp (Caridea: Bresiliidae): evidence from mitochondrial cytochrome oxidase subunit 1. Molecular Phylogenetics and Evolution 13, 244–254. Sharma, S., Hughes, J.M., 2009. Genetic structure and phylogeography of freshwater shrimps (Macrobrachium australiense and Macrobrachium tolmerum): the role of contemporary and historical events. Marine and Freshwater Research 60, 541–553. Shokitai, S., 1978. Larval development of interspecific hybrid between Macrobrachium asperulum and Macrobrachium shokitai from the Ryukyus. Bulletin of the Japanese Society of Scientific Fisheries 44 (11), 1187–1195. Sokal, R.R., Rohlf, F.J., 1995. Biometry: The Principles and Practice of Statistics in Biological Research, 3rd edition. W.H. Freeman and Co., New York. Thorpe, R.S., 1976. Biometric analysis of geographic variation and racial affinities. Biological Reviews 51, 407–452. Trontelj, P., Machino, Y., Sket, B., 2004. Phylogenetic and phylogeographic relationships in the crayfish genus Austropotamobius inferred from mitochondrial CO1 gene sequences. Molecular Phylogenetics and Evolution 34, 212–226. Ward, R.D., Woodwark, M., Skibinski, D.O.F., 1994. A comparison of genetic diversity levels in marine, freshwater and anadromous fish. Journal of Fish Biology 44, 213–232. Wowor, D., Muthu, V., Meier, R., Balke, M., Cai, Y.X., Ng, P.K.L., 2009. Evolution of life history traits in Asian freshwater prawns of the genus Macrobrachium (Crustacea: Decapoda: Palaemonidae) based on multilocus molecular phylogenetic analysis. Molecular Phylogenetics and Evolution 52, 340–350.