Two New Species of the Genus Dictyota ( Phaeophyceae : Dictyotales ) from the Mexican Caribbean

During a molecular characterization of the genus Dictyota J. V. Lamouroux (Phaeophyceae, Dictyotales) along the Mexican coasts of the Gulf of Mexico and the Mexican Caribbean, using psbA and cox1 genes, it was found that two particular collections did not match with the sequences published for a total of 39 species of Dictyota. For each gene, both collections diverged in high-uncorrected “p” distance values with respect to other related species. This high genetic divergence was corroborated by morphological analysis. Hence, we proposed Dictyota mayae J. Lozano-Orozco & Sentíes and Dictyota pedrochei J. Lozano-Orozco & Sentíes as new species.


Materials and Methods
The specimens studied in the present study were collected at Punta Brava and Puerto Morelos, Quintana Roo, in March 2012, by J.G. Lozano-Orozco and A. Sentíes.Voucher samples were fixed in 4% formalin solution for morphological analysis.
The specimens were examined using a Leica stereomicroscope (Leica MZ 12.5, Wetzlar, Germany).Transverse hand sections in the apical, middle and basal regions of the thallus were made with a stainless steel razor blade under a dissecting scope.Photomicrographs were taken with an Olympus Vg-160 digital camera (Tokyo, Japan) coupled to a Nikon Eclipse E200 microscope (Tokyo, Japan).Voucher specimens were deposited at UAMIZ Herbarium (UAMIZ1231, UAMIZ1232, UAMIZ1233 and UAMIZ1234).
Total DNA was extracted from silica gel-preserved material using a Dneasy Mini Kit (Qiagen, Valencia, CA, USA) following the manufacturers' instructions.A total of 956 bp from the psbA gene were amplified using the psbA F and psbA R primers [16], and 672 bp were amplified for cox1 with Gaz1R and Gaz1F primers (adapted from [21]), using the AmpliTaq kit of the Applied Biosystems (Lincoln Ventre Drive Foster City, CA, USA).All PCR products were checked for their correct length by electrophoresis on 1% agarose gel.The PCR products were sent to Macrogen (Gasan-dong, Seoul, Korea) to be purified and sequenced.Alignments for psbA and cox1 sequences were constructed using MEGA version 5 [22].Collection sites and GenBank Accession Numbers of the specimens used in the phylogenetic analysis are provided in Table 1.
Phylogenetic relationships were inferred using a concatenated matrix (1703 nt) of two genes from 39 taxa (psbA = 1031 bp; cox1 = 672 bp; Table 1).Canistrocarpus crispatus (J.V. Lamouroux) De Paula & De Clerck, Dictyopteris polypodioides (A.P. De Candolle) J. V. Lamouroux, Rugulopteryx okamurae (E.Y. Dawson) I. K. Hwang, W. J. Lee & H. S. Kim and Scoresbyella profunda Womersley were included as outgroup taxa based on previous results obtained by [23].The intraspecific and interspecific divergence values obtained for psbA and cox1 were calculated using uncorrected "p" distances implemented in MEGA version 5 [22].The Bayesian phylogenetic analysis was inferred with MrBayes v.3.0 beta 4 software [24] using the general time-reversible model of nucleotide substitution with invariant sites and Gamma-distributed rates for the variables sites (GTR + I + G).This model was selected based on Maximum Likelihood (ML) ratio test implemented by the software ModelTest 0.1.1 [25].For the Bayesian analysis, five chains of the Markov chain Monte Carlo (one hot and four cold) were run, sampling one tree every 1000 generations for 5 × 10 6 generations starting with a random tree.Maximum likelihood analysis was performed using TOPALI v2 software [26] with the GTR + I + G model.The ML bootstrap analyses were conducted with 100 replicates.Support values [27] were computed, as implemented in TOPALI v2.

Distribution and habitat:
The species is known only from the type locality, growing epilithically at this locality exposed in the shallow sublittoral.

Molecular Analysis
A total of 39 sequences were analyzed (Table 1).The Maximum Likelihood and Bayesian trees obtained for each marker were similar (not shown).Additionally, a phylogenetic analysis was carried out combining the partial sequences of psbA and cox1 genes of the 39 correspondent taxa, analysis of ML and IB produced highly congruent trees, differing in some positions that had poor support clades, but where the two new species are located, both analyses produced excellent support clades (Figure 3).Both new species diverged in high-uncorrected p distances values from the rest of the Dictyota representatives (>0.8% psbA and >11% cox1), confirming that these two samples constitute new taxonomic entities, or evolutionary independent lineages.

Phylogenetic Analysis
Considering the problems with the identification from the majority of Dictyota species based solely on morphological, anatomical or reproductive characters, we performed a phylogenetic analysis based on psbA and cox1 partial sequences.Two clades representing two undescribed species become apparent.These taxa, Dictyota mayae and D. pedrochei, are clearly delineated by long and highly supported branches.The divergence of psbA and cox1 sequences within the new species and the other Dictyota species was high (>0.8%psbA and >11% cox1).We determine that are new taxonomic entities because the divergence values obtained in the psbA gene are within intraspecific (<1%) and interspecific limits (<7%) previously reported in the literature for Dictyotales [18] [29].

Morphological Comparison and Distribution
Table 2 shows character distribution for species previously reported, plus the two new species here proposed.The morphological comparison between D. mayae and the other species phylogenetically related reveal striking differences.One of the main features is that D. mediterranea (Schiffner) G. Furnari and D. fasciola (Roth) J. V. Lamouroux have a multilayered medullary layer, with no iridescence in coloration, while D. mayae has a single medullary layer, color in situ is brown, with clear iridescence.In addition, D. fasciola and D. mediterranea are distributed in the European Atlantic and Mediterranean Sea.In the case of D. pedrochei, it also has several discrepancies with D. acutiloba J. Agardh, a phylogenetically related species that has a spiraling thallus, with a height to 20 cm, tufts of hairs in centrally located irregular rows on both surfaces, while D. pedrochei has a flattened thallus, a height to 5 cm and, surface proliferation in the middle of thallus without hairs.Primarily, D. acutiloba has a greater height than that of D. pedrochei, which is from Mexican Caribbean while D. acutiloba is strictly from the Pacific Ocean.

Conclusion
In this study, we perform morphological and molecular analyses on several species belonging to the genus Dictyota with special reference to the Mexican Caribbean.Analyses of thallus construction and a concatenated matrix of two phylogenetic markers have been found successful in supporting our conclusion that D. mayae and D. pedrochei are two evolutionarily independent lineages, and consquently two different species for the science.The application of morphological and molecular tools is improving our understanding of the biodiversity in the Mexican Caribbean, and in particular, to develop a model to have a better understanding of global diversity of Dictyota.

Figure 3 .
Figure 3. Phylogenetic relationship of Dictyota representatives based on concatenated Bayesian analysis of psbA and cox1 DNA sequences.The GTR + I + G evolutionary model was used in the Bayesian Analysis, selected by a maximum-likelihood radio test.Bayesian posterior probability (right) and ML bootstrap (left) values are indicated at the node.The localities are from the psbA sequences (right) and cox1 (left); other dates are shown inTable 1.

Table 1 .
Taxa used in this study for phylogenetic analysis.