The challenges of delimiting and identifying marine invertebrate species impede estimations of true biodiversity. This is particularly true in the case of gorgonian diversity, in which only classical morphological characters (e.g. branching pattern, size and colouration of the colony and sclerites, etc.), which can be homoplastic and continuous, have been used. In this study, using an integrative taxonomic approach, we analysed two morphs initially considered as two eco-typical variants of
Las dificultades existentes en la delimitación e identificación de especies de invertebrados marinos, impiden estimar la verdadera biodiversidad. Esto es particularmente observable en el caso de estudios sobre la diversidad de gorgonias, en los que tradicionalmente sólo se han utilizado caracteres morfológicos (por ejemplo, patrón de ramificación, tamaño y coloración de la colonia y escleritos, etc.) que pueden ser homoplásicos y continuos. En este estudio, utilizando un enfoque basado en una taxonomía integradora, se analizaron dos morfologías inicialmente consideradas como dos variantes eco-típicas de la especie
In the last decade, several studies have highlighted the difficulties of identifying marine species, especially invertebrates, using only morphological characters (
Understanding species boundaries in corals, particularly in octocorals, is a challenge for several fields of study, including taxonomy, evolutionary biology, life history and ecology (
Although molecular studies of octocorals have been performed, resolution at the species level is not always observed, such as in the genera
Methodological advances in morphological analysis, such as the use of morphometric statistics, can provide additional characteristics regarding the shape and structure of organisms (
In other ecosystems, combining analyses has provided a stronger evaluation of species identification (
Using such an approach, we investigated two Ecuadorian morphotypes of the genus
Here, we report our analyses of the morphological and genetic variation of two
To account for the high range of size variability, colonies were collected according to four size classes, determined by the distance between the holdfast and the most distant branch tip (class 1, ≤70 mm; class 2, between 71 and 140 mm; class 3, between 141 and 210 mm; and class 4, >211 mm).
Photographs of the sampled specimens were first taken underwater (
Collected specimens were deposited in the Museo Ecuatoriano de Ciencias Naturales (MECN), the octocoral reference collection of the research group “Biodiversidad y Ecología de Invertebrados Marinos” at the University of Seville (BEIM), the Museo Nacional de Ciencias Naturales in Madrid (MNCN-CSIC) or the Museu de Ciénces Naturals in Barcelona (MZB).
Colony fragments were prepared for scanning electron microscopy (SEM) according to standard methods (
For each colony, the total area and total area without gaps were measured (
The following measurements were taken from each colony: maximum height, maximum width, and average and maximum lengths of the primary branches. The number of ramifications and the number of branches of each order were also counted.
The following colony parameters were measured and calculated: mean width (mean of three measurements, taken at equidistant positions at a right angle to the height) (
As two highly collinear variables contain the same information and would be redundant for the purpose of analysis (
Genomic DNA was extracted from 20-30 mg of tissue using the DNeasy extraction Kit (Qiagen, Inc.), according to the manufacturer′s protocol. Amplifications were carried out in 50 µL final volume reactions containing 5 µL of 10× buffer (containing 10× 2 mM MgCl2), 1 µL dNTPs mix (10 mM), 0.8 µL of each primer (10 µM), 0.5 µL of Taq DNA polymerase (5U/µL) (Biotools) and 2 µL of genomic DNA. Cycling parameters and primer combinations used for each marker are given in
Primer | Code* | Sequence | Reference | PCR protocol | |
---|---|---|---|---|---|
Cox | COII8068F | 1 | 5´-CCATAACAGGACTAGCAGCATC-3´ | 58°C 45:60:60 ×40 | |
COI- Gorg2-F2 | 2 | 5´-GATTCGGAAATTGGTTTGTG-3´ | Present paper | ||
COIOCTR | 3 | 5´-ATCATAGCATAGACCATACC-3´ | |||
COI-Gorg1-R3 | 4 | 5´ AGAGAAGGTGGTAATAACCAGAAA-3´ | Present paper | ||
mtMutS | ND42599F | 5 | 5´-GCCATTATGGTTAACTATTAC-3´ | 58°C 90:90:60 ×35 | |
MUT3458R | 6 | 5´-TSGAGCAAAAGCCACTCC-3´ | |||
ITSs | ITS 2.1 | 7 | 5´-CGTAGGTGAACCTGCGGAAGGATC-3´ | 56°C 60:90:60 ×35 | |
ITS 2.2 | 8 | 5´-CCTGGTTAGTTTCTTTTCCTCCGC-3´ | |||
28S rRNA | 28S-Far | 9 | 5´-CACGAGACCGATAGCGAA CAAGTA-3´ | 50°C 90:90:60 ×30 | |
28S-Rar | 10 | 5´-TCATTTCGACCC TAAGACCTC-3´ | |||
28S-R3 | 11 | 5´-ACTGCATRTATGAACTCCA-3´ | Present paper |
The molecular data matrix consists of sequences from species reported here and from other recently sequenced species including
Species | Igr + COI | mtMutS | ITSs | 28S |
---|---|---|---|---|
GQ342423 | GQ342499 | EU043125 | JX203712 | |
KX721173 | KX721192 | KX721211 | KX721230 | |
Eugorgia daniana (1) | KX721188 | KX721207 | KX721226 | KX721245 |
KX721189 | KX721208 | KX721227 | KX721246 | |
KX721176 | KX721195 | KX721214 | KX721233 | |
KX721182 | KX721201 | KX721220 | KX721239 | |
KX721183 | KX721202 | KX721221 | KX721240 | |
KX721184 | KX721203 | KX721222 | KX721241 | |
KX721186 | KX721205 | KX721224 | KX721243 | |
KX721187 | KX721206 | KX721225 | KX721244 | |
KX721177 | KX721196 | KX721215 | KX721234 | |
KX721178 | KX721197 | KX721216 | KX721235 | |
KX721179 | KX721198 | KX721217 | KX721236 | |
KX721180 | KX721199 | KX721218 | KX721237 | |
KX721181 | KX721200 | KX721219 | KX721238 | |
KX721185 | KX721204 | KX721223 | KX721242 | |
KX721190 | KX721209 | KX721228 | KX127247 | |
KX721174 | KX721193 | KX721212 | KX721231 | |
KX721191 | KX721210 | KX721229 | KX721248 | |
KX721175 | KX721194 | KX721213 | KX721232 |
The model that best fit for nucleotide evolution for each final alignment was determined with jModelTest (
ML and MP supports were determined through 1000 bootstrap replicates (bootstrap values = bv). Bayesian inference of phylogenetic relationships was performed in MrBayes 3.1 (
Based on the evidence provided by the study of the differentiation of two
Family Gorgoniidae Lamouroux, 1812
Genus
(
For a complete description and list of the materials of
Nine morphological characteristics were selected: maximum height, maximum length of the primary branches to maximum height ratio, order of colony, bifurcation ratio, tributary to source ratio of secondary order branches, main thickness of the primary order branches, angle formed between primary and secondary order branches, angle formed between secondary and tertiary order branches, and angle formed by basal branches. There were significant differences in the multivariate structure between the species,
df | SS | MS | Pseudo-F | P(perm) | |
---|---|---|---|---|---|
Morph | 1 | 39.80 | 39.80 | 5.67 | <0.01 |
Size | 3 | 129.5 | 43.16 | 6.15 | <0.01 |
Morph × size | 3 | 29.79 | 9.93 | 1.41 | 0.10 |
Residual | 69 | 484.22 | 7.01 | ||
Total | 76 | 684 |
One matrix was prepared with the concatenated data from the four marker alignments: mtMutS+Cox+ITSs+28S (16 specimens, 3353 characters). New sequences were deposited in GenBank (
The reconstruction based on the data obtained from the four concatenated genes (
The morphological and morphometric results presented here, which showed significant differences in colony shape and branching, support the existence of two distinct species,
Previous morphological studies based on a limited set of characters, including
Moreover,
Most of the morphometric characters considered in this study were not noted in previous descriptions and revisions. In addition, our study shows that the differences between
The significant differences observed in the morphometric and morphological analyses were not completely consistent with our molecular results, in which the relationship between
Although for some molecular phylogenies of corals, hybridization processes have been used to explain a lack of resolution, polyphyly or paraphyly (
Evolutionary status and the relatedness of species, and the traits used to identify them, may not have a direct positive relationship (
Overall, the differentiation reported here between the new species
Of the approximately 28
This research was partially supported by a grant from the Spanish Ministry of Economy and Competitiveness (CTM2014-57949-R). Our appreciation goes to Santiago Villamarín (Museo Ecuatoriano de Ciencias Naturales), Machalilla National Park and the Ministerio del Ambiente of Ecuador (Manabí) for participation and collection permits (Nº 016-RM-DPM-MA). We thank Michel Guerrero and his team (Exploramar Diving) for his special interest and collaboration. Melinda Modrell carefully reviewed the language. Special thanks to Micaela Peña for her strong support.