Molecular analyses

Molecular analyses were based on two mitochondrial genes, a fragment DNA of the 16S rRNA (16S) and the barcode region of the Cytochrome C Oxidase subunit I (COI). Both genes are widely used in phylogenetic studies of many invertebrates and decapod crustaceans [22–25]. More specifically for decapod crustaceans, both genes have been used in recent barcoding projects [26–28] and U.S. National Science Foundation Decapod Tree of Life phylogenetics studies [29–31] to delimit species boundaries and to clarify the evolutionary relationships among decapod crustaceans.

DNA extraction, amplification, and sequencing protocols followed Schubart et al. [32] with modifications according to Mantelatto et al. [33] and Robles et al. [34]. Total genomic DNA was extracted from muscle tissue of the chelipeds. The tissue was incubated for 48h in 600μL of lysis buffer at 55°C, with 200μL of proteinase K (PK); protein was separated by addition of 200μL of 7.5M ammonium acetate prior to centrifugation. DNA was precipitated by addition of 600μL of isopropanol cooled to the supernatant and then centrifuged; the resultant pellet was washed with ethanol 70%, centrifuged, dried and resuspended in 10–20μL TE buffer.

An approximately 550bp region of the 16S rDNA gene and 650bp region of the COI gene were amplified by polymerase chain reaction (thermal cycles: initial denaturing for 5min at 94–95°C; annealing for 35–40 cycles: 45sec at 95°C, 45sec at 42–48°C, 1min at 72°C; final extension of 5min at 72°C) with universal primers to invertebrates 16Sbr (5’-CCG GTC TGA ACT CAG ATC AC-3’) and 16Sar (5’-CGC CTG TTT ATC AAA AAC AT-3’) [35]; COH6 (5’-TAD ACT TCD GGR TGD CCA AAR AAY CA-3’) and COL6b (5’-ACA AAA TCA TAA AGA TAT YGG-3’) [36].

PCR products were purified using a SureClean Plus kit (following the vendor’s protocols) and sequenced using the ABI Big Dye^® Terminator Mix in an ABI Prism 3100 Genetic Analyzer^® following Applied Biosystems protocols. The sequences obtained were confirmed by sequencing both strands; consensus sequences were obtained using BioEdit version 7.0.5 [37] from the two complementary sequences. Consensus sequences of the 16S and COI genes were aligned using ClustalW [38] as implemented in BioEdit [37], with default parameters. The COI sequences were checked for the presence of stop codons. All sequences were submitted to GenBank.

The concatenated analyses were conducted based on a total of 1091bp (606 for the COI and 485 for the 16S genes, excluding the primer regions). Alignment of both gene sequences was unambiguous and the ILD test showed no significant incongruence. After confirming that the two genes have the same evolutionary history, the best-fitting model for sequence evolution of the combined COI and 16S was determined by JModelTest 2.1.4 [39], selected by the AIC (Akaike information criterion) method. This information criterion indicated the TPM1uf+I+G as the best-fit model of DNA sequence evolution, accounting for invariable positions and unequal rates of substitutions under a gamma distribution, with the nucleotide frequencies: A = 0.3626, C = 0.1266, G = 0.1618, and T = 0.3490; rates of nucleotide substitution A-C = 1.0000, A-G = 94.1738, A-T = 7.0694, C-G = 7.0694, C-T = 94.1738, G-T = 1.0000; proportion of invariable sites I = 0.6530; and gamma shape = 0.8590.

The Bayesian analysis (BAY) was performed with MrBayes 3.2.2 [40] with the parameters obtained from JModeltest. The search was run with four chains for 20,000,000 generations with trees being sampled every 10,000 generations.

Trace plots were visually inspected to assess convergence, mixing, and stationarity in Tracer v1.4. [41]. Once the split frequency in each analysis was 1% (reached well before 2 million generations = 200 trees), we found the maximum clade credibility tree (MCCT) using TreeAnnotator v1.5.4 [42] from the remaining 1800 saved trees. We obtained a 50% majority rule consensus tree using the same 1800 trees. Bayesian posterior probability [43]; values > 70% were shown on the resulting MCCT.

Maximum Likelihood analysis (ML) was performed with RAxML 7.0.4 [44], as implemented in CIPRES (Cyberinfrastructure for Phylogenetic Research). The model of evolution was the GTR+G+I, which is the default model for RAxML. The internal consistency of the branches was evaluated by the bootstrap method [45], and we selected the option to automatically determine the number of bootstraps to be run in RAxML. A total of 150 bootstrap pseudoreplicates were run, and confidence values > 50% were shown on the resulting trees.

Genetic distance analyses were applied in previous systematic studies of crustaceans and other animals [46–50]. Genetic distance calculations were performed using MEGA 5 [51], and two distance matrices were calculated using uncorrected distances (p-distance), based on COI and 16S sequences. We did not calculate a distance matrix using a model of evolution since it has been shown that using p-distance avoid over-parametrizing and there is no need to use complex distances measures when studying closely related sequences [52, 53]. To help assess intraspecific and interspecific genetic distances, two frequency histograms were constructed with pairs of COI and 16S sequences.