Population Genetics and Evolutionary Demography of Ucides Cordatus (Decapoda: Ocypodidae) Jose´ F
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Marine Ecology. ISSN 0173-9565 ORIGINAL ARTICLE Population genetics and evolutionary demography of Ucides cordatus (Decapoda: Ocypodidae) Jose´ F. Oliveira-Neto1,2, Marcio R. Pie1,2, Walter A. Boeger1,2, Antonio Ostrensky2,3 & Rafael A. Baggio1,2 1 Departamento de Zoologia, Universidade Federal do Parana´ , Curitiba, PR, Brazil 2 Grupo Integrado de Aqu¨ icultura e Estudos Ambientais, Universidade Federal do Parana´ , Curitiba, PR, Brazil 3 Departamento de Zootecnia, Universidade Federal do Parana´ , Curitiba, PR, Brazil Keywords Abstract Brachyura; land crab; mangrove; mismatch distribution; nested clade analysis; population Variation in a fragment of the mitochondrial control region was investigated to expansion; population genetics. assess the population history of the mangrove land crab, Ucides cordatus L., based on 100 individuals from five locations throughout the Brazilian coast. There Correspondence was considerable variability in the studied fragment, with every individual Marcio R. Pie, Departamento de Zoologia, showing a distinct haplotype. When regions of ambiguous alignment were Caixa Postal 19073, Universidade Federal do removed, 90 haplotypes were still recognized, of which only 1 was shared Parana´ , Curitiba, PR 81531-990, Brazil. E-mail: [email protected] among locations. There was no statistically significant evidence for geographical structure in the pattern of genetic variability based on a nested clade analysis, Accepted: 20 June 2007 even though the geographical distances among the farthest collection sites exceeded 3000 km. Moreover, the mismatch distribution of pairwise differences doi:10.1111/j.1439-0485.2007.00188.x showed a smooth unimodal distribution that is consistent with a recent popu- lation expansion of U. cordatus, either when each location was analysed in isolation or when all samples were combined. Interestingly, the estimates for the duration of such expansion vary latitudinally, suggesting that the expansion of the population of U. cordatus proceeded in a southward direction. These results indicate a possible relationship between the growth in the population of the mangrove land crab and the dynamics of mangrove ecosystems during the Quaternary. Pfeiler et al. 2005). However, there is still no comprehen- Problem sive picture of how physiographical and historical events The increasingly larger number of analytical and concep- have shaped the patterns of genetic diversity of marine tual tools for the study of genetic variability has revealed species since the beginning of the Quaternary. Such infor- a remarkable variety of patterns of gene flow and histor- mation would be instrumental, not only to understand ical demography of marine organisms (Graves 1998; Shaw the diversification process of marine taxa, but also to et al. 1999; Weber & Levy 2000; Knutsen et al. 2003; establish conservation strategies that maintain the long- Roman & Palumbi 2004; Bay et al. 2006; Santos et al. term evolutionary potential of these species (Palumbi 2006). Even among closely related species, one can find 2004). drastically different degrees of geographical structure and A marine crustacean of particular importance in Brazil- genetic diversity, as recently observed in two closely rela- ian mangrove areas is the mangrove land crab Ucides ted penaeid shrimp species (McMillen-Jackson & Bert cordatus L. It is considered a keystone species in man- 2003), as well as several species of Callinectes crabs groves of the western Atlantic for its role in processing (Weber & Levy 2000; McMillen-Jackson & Bert 2004; decaying plant material and nutrient cycling in those 460 Marine Ecology 28 (2007) 460–469 ª 2007 The Authors. Journal compilation ª 2007 Blackwell Publishing Ltd Oliveira-Neto, Pie, Boeger, Ostrensky & Baggio Population genetics of Ucides cordatus environments (Nordhaus et al. 2006). In addition, it is (5¢-GCT AYC CTT TTA AAT CAG GCA C-3¢) were among the most important fishery resources in northeast- used for the amplification of an 1.6 kb fragment inclu- ern Brazil, particularly among underprivileged coastal ding the complete CR of U. cordatus (Oliveira-Neto communities (Diele et al. 2005). A combination of over- et al. 2007). The CR is a non-coding fragment of the exploitation (Ministe´rio do Meio Ambiente 2004; Amaral mitochondrial genome. In crustaceans, the CR is usually & Jablonski 2005) and an emerging infectious disease (the flanked on its 5¢ end by the 12S ribosomal gene and on Lethargic Crab Disease, Boeger et al. 2005) has caused its 3¢ end by the isoleucine-tRNA. Each 25 ll PCR severe depressions in natural stocks of U. cordatus (Alves included the following final concentrations: 6 mm of )1 & Nishida 2003), leading to an urgent necessity to MgCl2, 0.25 mm of each dNTP, 0.1 U ll of Taq po- develop efficient management strategies to maintain the lymerase, 1X buffer, 2 lm of each primer, and ) genetic integrity of local populations. The first genetic 1.2 ng ll 1 of template DNA. Thermocycling conditions study of this species was carried out by Oliveira-Neto included an initial denaturation at 95 °C for 2 min, fol- et al. (2007) using RAPD and PCR-RFLP of the mitoch- lowed by 35 cycles at 95 °C for 20 s, 56 °C for 30 s, ondrial control region (CR) and showed no evidence for and 72 °C for 90 s, and a final extension at 72 °C for genetic structure in this species. However, further studies 2 min. A 2 ll aliquot of each PCR product was electro- using sequence data and a broader geographical range are phoresed in a 1.5% agarose gel, stained with ethidium necessary to establish these findings firmly. bromide and visualized under UV light. Successfully Changes in the size of a population tend to leave amplified products were purified using a MinElute kit recognizable signatures in its patterns of genetic variabil- (Qiagen). Sequencing reactions were carried out using ity. In particular, the distribution of pairwise sequence internal primers (DL.USSA.R1: 5¢-GGTTAGAGAGA differences in a sample (known simply as the mismatch AGGTTAGAGGAC-3¢, and DL.USSA.F1: 5¢-GTATA distribution) can contain information on the history of a ACCGCGAATGCTGGCAC-3¢), which generate a frag- population (Rogers & Harpending 1992). Given that the ment that could be reliably sequenced in a single reac- genealogy of a population of constant size is expected to tion, including 112 bp of the 5¢-flanking 12S gene and have long deep branches, mutations occurring along these approximately 400 bp into the CR (actual size varied branches will be shared by several lineages, resulting in an due to indels). Cycle sequencing in 10 ll solutions ) irregular or ragged mismatch distribution. Conversely, the included the following final concentrations: 5 ng ll 1 of genealogy of a population that has substantially grown in template DNA, 0.16 lm of primer, 0.15X of reaction size has long terminal branches, and the mutations that buffer, and 0.5 ll of BigDye (Applied Biosystems). The have occurred along these branches will be specific to sin- final product was purified using Sephadex G50 and gle lineages. Under these conditions, a unimodal mis- processed on a ABI3130 automatic sequencer. Forward match distribution is expected, whose mean, under an and reverse strands were reconciled using the Staden infinite-sites mutation model, increases as a function of package (Staden 1996). Sequences were aligned using the time elapsed after population growth. Mismatch dis- Clustal W (Thompson et al. 1994), as implemented in tribution methods are used in the present study to infer BioEdit (Hall 1999). Regions with ambiguous alignment the population history of the mangrove land crab, were omitted from further analyses. U. cordatus, through the investigation of the patterns of The existence of geographical structure in the pattern variation in a fragment of the mitochondrial DNA CR of genetic variability among populations of U. cordatus among locations along the Brazilian coast. The observed was tested using nested clade analysis (NCA), as imple- results are consistent with the expansion of U. cordatus mented in the program GeoDis (Posada et al. 2000) populations, which seems to have occurred from the according to the methods of Templeton et al. (1995). northernmost locations into higher latitudes. This program calculates the clade distances Dc (average distance between the location of the members of the clade and the geographical center of the clade), and the nested Material and Methods clade distances Dn (the average spatial distance between Samples of U. cordatus were obtained from several loca- the members of each clade and the geographical centre of tions throughout the Brazilian coast (Fig. 1). Muscle tis- the entire nesting clade). Additionally, the measures of sue from one of the pereiopods was removed, preserved average distance between tip and interior clades within in EDTA-DMSO buffer (Seutin et al. 1991), and main- the nested group (Int-Tip)c, and the tip to interior distance tained at )20 °C. Genomic DNA was extracted using for the nesting clade (Int-Tip)n are estimated. The assess- the DNeasy kit (Qiagen) according to the manufacturer’s ment of whether any of these distances is significantly instructions. The primers 12SUCAF3 (5¢-CCA GTA NRC smaller or larger than expected by chance was carried out CTA CTA TGT TAC GAC TTA T-3¢) and ILEUCAR3 by 10,000 permutation resamplings. Interpretation of the Marine Ecology 28 (2007) 460–469 ª 2007 The Authors. Journal compilation ª 2007 Blackwell Publishing Ltd 461 Population genetics of Ucides cordatus Oliveira-Neto, Pie, Boeger, Ostrensky & Baggio Fig. 1. Map of the coast of Brazil indicating the sampled locations. Detailed coordinates of each location can be obtained from the authors upon request. results followed the method given in Templeton et al. ized nonlinear least-square approach developed by (1995). Schneider & Excoffier (1999). Also, the degree of approxi- Inferences on past demographic history were assessed mation between the observed mismatch distribution and using the mismatch distribution analysis (Slatkin & Hud- that expected under population growth was tested using son 1991; Rogers & Harpending 1992).