Figure 1.
External morphology of Mexican Pacific Aplysina species.
A,B) Aplysina clathrata sp. nov.; C,D) Aplysina revillagigedi sp. nov.; E,F) Aplysina gerardogreeni.
Figure 2.
Skeletal characteristics of Mexican Pacific Aplysina species.
A–E) A. clathrata sp. nov.; A) Regular tridimensional skeletal reticulation of a fistular proyection; B,C,D) Detail of skeleton sponging fibers with nodular pith and short protuberances (showing by arrows); E) Transversal view of fibers showing by arrows; F–H) A. revillagigedi sp. nov.; F) Tridimensional skeletal reticulation at deep choanosome; G) Dentritic-like terminal skeletal fibers; H) Transversal view of fibers showing by arrows; I,J) A. gerardogreeni; I) Regular tridimentional skeletal reticulation; J) Transversal view of fibers showing by arrows.
Figure 3.
Sampling localities and distribution of Aplysina species along the Mexican Pacific Ocean.
Numbers correspond to different species: (1) Aplysina clathrata sp. nov.; (2) Aplysina revillagigedi sp. nov.; (3) Aplysina gerardogreeni.
Table 1.
Comparative data of external morphology, skeletal characteristics and distribution of Aplysina species from Eastern Pacific and Atlantic Oceans and the Mediterranean Sea.
Figure 4.
Neighbor-Joining phylogenetic reconstruction of Eastern Pacific and Caribbean species of the genus Aplysina.
Numbers associated to each branch represent: NJ/MP bootstrap support values/Bayesian posterior probabilities (%). (-) indicates that a particular branch was not present in the MP or Bayesian reconstruction.
Figure 5.
Results of the infrared spectroscopy of purified skeletons.
Aplysina fulva; A) A. gerardogreeni; B) A. clathrata sp. nov.; C) A. revillagigedi sp. nov.; D) Suberea azteca. Apysina fulva, and Suberea azteca (unpublished data) are only included for comparative purposes.
Figure 6.
Demineralized and purified skeletal fibres of Aplysina revillagigedi sp. nov.
A) Light microscopy image; B) Show intensive fluorescence after Calcofluor White staining for chitin. The light exposure time for fluorescence microscopy was 1/1000 s.
Table 2.
Accession numbers of the specimens sequences, vouchers and DNA sequences analyzed.
Figure 7.
Diagnostic nucleotides and groups of nucleotides following Davis and Nixon [48] for three species of Aplysina from the Mexican Pacific.
Single-nucleotide pure diagnostic characters are individually color-coded for each species (green: A. gerardogreeni, blue: A. revillagigedi, and yellow: A. clathrata); additional composite diagnostic combinations are indicated for A. revillagigedi (orange) and A. clathrata (gray). ITS1-5.8S-ITS2, nuclear ribosomal DNA; COI, mitochondrial cytochrome oxidase subunit I. Nucleotide residues refer to the individual alignments of ITS1-5.8S-ITS2 and COI sequences (GenBank accessions: ITS1-5.8S-ITS2 rDNA JN596955–58 and COI mtDNA JQ437578–80). Nucleotide 101 is ITS1 and the rest are ITS2.
Table 3.
Pairwise ITS1-5.8S-ITS2 rDNA genetic divergence (% uncorrected p-distance) between Aplysina species, from Eastern Pacific and Caribbean.
Figure 8.
Delineation of new Aplysina species from the Mexican Pacific under the framework of the taxonomic circle [12].
A) The first hypothesis consists in differentiating the new species A. clathrata sp. nov. and A. revillagigedi sp. nov. from A. gerardogreeni, based on an initial morphological distinction. For this, ecological and structural biochemical evidence per se are not conclusive to break-out of the circle. Geography could allow breaking-out the circle for A. revillagigedi sp. nov. (dashed arrow), an insular endemic, but not for both. However the integration of the molecular data in the form of diagnostic characters as well as quantitative divergence and phylogenetic analyses provide congruent information with the rest allowing to breakout of the circle; B) The second hypothesis consists in differentiating the two new species. For this geographical distribution (continental vs. insular) could help to break out of the circle (dashed arrows) but structural biochemistry remains inconclusive. However, the addition of molecular evidence indicating the genetic distinction and diagnostic characters in the nuclear and mitochondrial genomes provide the unequivocal evidence of reproductive isolation, allowing breaking out of the circle.