Integrative taxonomy and molecular phylogeny of genus Aplysina (Demospongiae: Verongida) from Mexican Pacific

Abstract

Integrative taxonomy provides a major approximation to species delimitation based on integration of different perspectives (e.g. morphology, biochemistry and DNA sequences). The aim of this study was to assess the relationships and boundaries among Eastern Pacific Aplysina species using morphological, biochemical and molecular data. For this, a collection of sponges of the genus Aplysina from the Mexican Pacific was studied on the basis of their morphological, chemical (chitin composition), and molecular markers (mitochondrial COI and nuclear ribosomal rDNA: ITS1-5.8-ITS2). Three morphological species were identified, two of which are new to science. A. clathrata sp. nov. is a yellow to yellow-reddish or -brownish sponge, characterized by external clathrate-like morphology; A. revillagigedi sp. nov. is a lemon yellow to green, cushion-shaped sometimes lobate sponge, characterized by conspicuous oscules, which are slightly elevated and usually linearly distributed on rims; and A. gerardogreeni a known species distributed along the Mexican Pacific coast. Chitin was identified as the main structural component within skeletons of the three species using FTIR, confirming that it is shared among Verongida sponges. Morphological differences were confirmed by DNA sequences from nuclear ITS1-5.8-ITS2. Mitochondrial COI sequences showed extremely low but diagnostic variability for Aplysina revillagigedi sp. nov., thus our results corroborate that COI has limited power for DNA-barcoding of sponges and should be complemented with other markers (e.g. rDNA). Phylogenetic analyses of Aplysina sequences from the Eastern Pacific and Caribbean, resolved two allopatric and reciprocally monophyletic groups for each region. Eastern Pacific species were grouped in general accordance with the taxonomic hypothesis based on morphological characters. An identification key of Eastern Pacific Aplysina species is presented. Our results constitute one of the first approximations to integrative taxonomy, phylogeny and evolutionary biogeography of Eastern Pacific marine sponges; an approach that will significantly contribute to our better understanding of their diversity and evolutionary history.

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.

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.

Figure 7. Diagnostic nucleotides and groups of nucleotides following Davis and Nixon 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.

Figure 8. Delineation of new Aplysina species from the Mexican Pacific under the framework of the taxonomic circle .

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.