Leptuca thayeri (Rathbun, 1900)

Marochi, Murilo Zanetti, Tangerina, Marcelo Marucci Pereira, Rodrigues, Renata de Oliveira, Laurenzano, Claudia, Vilegas, Wagner, Costa, Tânia M. & Schubart, Christoph D., 2022, Phylogeographic Structure within the Fiddler Crabs and (Brachyura, Ocypodidae) along the Tropical West Atlantic., Zoological Studies 61 (67), pp. 1-15 : 3-5

Sampling of Leptuca thayeri and Uca maracoani

Leptuca thayeri specimens were collected from three Brazilian populations in the states of Pará (Marudá), Bahia (Acuípe), and São Paulo (Bertioga). In addition, 11 specimens from Jamaica (Priory, St. Ann) and the Dominican Republic (Luperón). Uca maracoani specimens were collected from one Brazilian population in Pará (Marudá), and one population in the Dominican Republic (Sánchez mangroves) ( Table 1) ( Fig. 1 ). All individuals sampled were deposited into the Zoologische Staatssammlung München ( Table 1).

DNA extraction, amplification, and sequencing

A total of 45 individuals of L. thayeri and 20 individuals of U. maracoani from five and two sample sites, respectively, were used for genetic analyses ( Tables 2 and 3). Genomic DNA was extracted from muscle tissue of pereiopods using the Puregene (Gentra Systems) buffer system method. For both species, DNA amplification from the mitochondrial gene cytochrome oxidase, subunit I (Cox1) was carried out by polymerase chain reaction (PCR) (40 cycles: 45 sec 94°C/1 min 48°C/75 sec 72°C denaturing/annealing/elongation temperatures). For L. thayeri and U. maracoani an 857 base pair region was amplified using the primers COL1b 5'-CCW GCT GGD GGW GGD GAY CC-3' and COH16 5'-CAT YWT TCT GCC ATT TTA GA- 3', and a shorter region of 640 base pairs using the primers COL1b 5'-CCW GCT GGD GGW GGD GAY CC-3' and COH1b 5'-TGT ATA RGC RTC TGG RTA RTC-3' ( Schubart 2009). This was necessary, because not all of the older specimens allowed to amplify the longer fragment, and in consequence two different datasets were evaluated, one maximizing the number of individuals (but with a short alignment), the other one maximizing the length of the alignment and exploring new variable positions (but with fewer representatives). Amplification results were checked by running 4 μl of PCR product on 1.5% TBE agarose gel electrophoresis. PCR products were outsourced for sequencing with the primer COL1b to Macrogen Europe, Inc. (Amsterdam, the Netherlands). Obtained sequences were edited in Chromas Lite 3.01 (Technelysium Pty Ltd 2005) and manually aligned with BioEdit 5.0 ( Hall 1999). Primer sequences and adjacent regions were omitted, resulting in an alignment of 826 or 609 base pairs for L. thayeri and 825 or 606 base pairs for U. maracoani . The software Artemis ( Rutherford et al. 2000) was used to rule out the presence of stop codons, which could indicate the presence of pseudogenes. Sequences were submitted to GenBank ( Table 1).

Genetic data analyses

The number of haplotypes, and haplotype (h) and nucleotide (π) diversities were calculated in DnaSP v5 ( Librado and Rozas 2009). To assess levels of genetic differentiation among populations, pairwise Φ ST values ( Weir and Cockerham 1984; Loh et al. 2001) were calculated with Arlequin ver. 3.11 ( Excoffier et al. 2005). A statistical parsimony network was constructed with PopArt ( Polzin and Daneshmand 2003; Leigh and Bryant 2015).

The variance between tested groups was assessed by Analyses of Molecular Variance (AMOVA), using Arlequin ver. 3.11. For L. thayeri two populations from Caribbean islands ( Jamaica and Dominican Republic) and three from Brazil (Pará, Bahia and São Paulo) were compared with the shorter DNA alignment (609 bp). The longer alignment (826 bp) could only be used to compare the northernmost Brazilian population (Pará) with the other northeast and southeast Brazilian populations (Bahia and São Paulo). For U. maracoani , one population from the Caribbean (Hispaniola) and one new one from Brazil (Pará) were compared to a relatively large dataset from GenBank (KF666951- KF666995), including Brazilian populations from Amapá, Maranhão, Ceará, Pernambuco, Bahia, Espírito Santo, Rio de Janeiro and Paraná ( Wieman et al. 2014).

To examine the population history and to evaluate whether the populations follow the neutrality model at the sampling sites, Tajima’s D, Fu’s Fs, and mismatch distribution analyses ( Tajima 1989; Fu 1997; Schneider and Excoffier 1999) were carried out using the Arlequin ver. 3.11 software. To test for significant restriction of gene flow, we used a non-parametric permutation procedure ( Excoffier et al. 1992), incorporating 10,000 permutations.

Figures

Fig. 1. Sample sites of Leptuca thayeri (black dots) and Uca maracoani (grey dots) populations from throughout the West Atlantic: Luperón & Sánchez from the Dominican Republic; Priory (St. Ann) from Jamaica; Marudá (Pará), Acuípe (Bahia), Bertioga (São Paulo) from Brazil. Grey squares correspond to localities sampled by Wieman et al. (2014), with sequences of U. maracoani in GenBank.