OBJECTIVES AN ASSESSMENT OF THE DIVERSITY ...

AN ASSESSMENT OF THE DIVERSITY OF ZOANTHARIANS FROM ECUADOR USING AN INTEGRATIVE APPROACH Karla B. Jaramillo1,2, Paul O.Guillen1,2, Grace McCormack2, Jenny Rodriguez1, Olivier P. Thomas2, Frederic Sinniger3 1. Escuela Superior Politécnica del Litoral, ESPOL. Centro Nacional de Acuacultura e Investigaciones Marinas, CENAIM. Km 30.5 via Perimetral, Guayaquil, Ecuador. 2. National University of Ireland Galway, University Road, Galway, Ireland. 3. Tropical Biosphere Research Center, University of the Ryukyus, Sesoko Island - Okinawa, Japan.

INTRODUCTION

OBJECTIVES

- The taxonomy of zoantharians is highly challenging due to a lack of clear morphological characters; although molecular techniques have helped to clarify high level-taxonomic relationships within the order. - However, many issues remain at species level due to the limited resolution of DNA markers commonly used. - Metabolomics approach showed promise to help distinguish closely related marine invertebrates like sponges1 or even morphotypes for zoantharians2. - The Eastern Equatorial Pacific is one of the most poorly studied area for zoantharians diversity (Figs. 1 and 2).

- Use a combined morphological and molecular approach to describe and classify zoantharians species of El Pelado marine protected area (Fig.1).

STUDY AREA

- Assess the potential of metabolomics to help identifying and characterizing zoantharian species.

400 m 300 km

Fig. 1. Map of El Pelado Marine Protected Area

Fig. 2. Map of Ecuadorian coast

METHODS Morphological observation

Molecular analyses Fixation in 95% EtOH

DNA Extraction of small subsamples of fixed tissues

Amplification/sequencing of mitochondrial 16S, COI and nuclear 18S, ITS

Sequences analyses (BioEdit, PhyML softwares)

Chemical analyses

Sampling

6 replicates of each species

RESULTS

Freeze dried samples

Metabolites extraction DCM/MeOH 1:1

Fractionation by C18 SPE with H2O, MeOH, Dichloromethane

El Pelado - Ecuador zoantharian diversity

Phylogenetic analyses

Methanol fractions were analyzed by UHPLC-Q ToF

Data analyses (msconvert, R/XMS, MetaboAnalyst softwares)

Metabolomics analyses - Metabolomic profiles were both consistent between all replicates within a species and distinct between species, despite specimens being collected at different times, depths and locations.

- In El Pelado Marine Protected Area, zoantharians are important components of the benthic fauna. - Several species found at El Pelado MPA were reported from the Galapagos Islands3,4 and Machalilla National Park5. a b Epizoanthus spp. 100

Antipathozoanthus spp. (a) “Parazoanthus” puertoricense

97

- According to metabolomics data, the undescribed species might be related to Terrazoanthus from a taxonomic point of view.

Umimayanthus spp.

Antipathozoanthus hickmani Parazoanthus darwini

c

(c)

d

Parazoanthus spp.

Fig. 5. Chromatographic profile of one replicate of Zoanthus sp1

EP410825 (b)

100 98

Savalia spp.

Hydrozoanthus spp.

Antipathozoanthus hickmani Parazoanthus darwini Terrazoanthus onoi Undescribed specie Zoanthus sp. 1 Zoanthus sp. 2

6 0

82 100

Zoanthus sp1

Terrazoanthus onoi

e

EP110828 (c) Terrazoanthus spp.

f

4 0

98

(g)

(b)

(a)

99

0.02

88

Palythoa spp. (f)

Zoanthus sp2

2 0 0

(d)

Palythoa cf. mutuki

g

-20

96

SP110819 (d) 100 Zoanthus spp. 87 AY111202 (e) Isaurus spp.

(e)

?

-40

91

PC 2 (16.3%)

89

-40

Fig. 3. Maximum Likelihood phylogenetic tree based on cytochrome c oxidase subunit I. Ecuadorian samples are indicated in red. Stars indicate predicted position based on morphological identification. Bootstrap support values >80% are indicated by the nodes.

Undescribed species

-20

0 PC 1 (24.7%)

20

40

60

Fig. 6. Scores plot between the most significant PCs. The explained variances are shown in brackets.

Fig. 4. In situ photographs of the 7 species found at El Pelado MPA. Scale above the names indicate approximately 1 cm.

25

20

15

10

5

Terrazoanthus onoi-5 Terrazoanthus onoi-2 Terrazoanthus onoi-6 Terrazoanthus onoi-4 Terrazoanthus onoi-3 Terrazoanthus onoi-2 UndSpec-4 UndSpec-2 UndSpec-3 UndSpec-1 UndSpec-6 UndSpec-5 Parazoanthus darwini-4 Parazoanthus darwini-3 Parazoanthus darwini-1 Parazoanthus darwini-6 Parazoanthus darwini-2 Parazoanthus darwini-5 Antipathozoanthus hickmani-4 Antipathozoanthus hickmani-2 Antipathozoanthus hickmani-3 Antipathozoanthus hickmani-1 Antipathozoanthus hickmani-6 Antipathozoanthus hickmani-5 Zoanthus sp1-6 Zoanthus sp1-3 Zoanthus sp1-4 Zoanthus sp1-1 Zoanthus sp1-5 Zoanthus sp1-2 Zoanthus sp2-3 Zoanthus sp2-2 Zoanthus sp2-4 Zoanthus sp2-6 Zoanthus sp2-5 Zoanthus sp2-1

0

Fig. 7. Hierarchical Clustering Dendrogram of the metabolomic data obtained with Pearson’s distances and Ward’s linkage algorythm.

DISCUSSION

PERSPECTIVES

- Despite its small geographical area, El Pelado MPA has a similar or even higher zoantharian diversity than the whole Galapagos region. - The results of metabolomics analyses performed on 6 species strongly support that this approach is useful as a complementary tool to morphological and molecular taxonomy of zoantharians. - Using the metabolomics approach, the undescribed species might be related to Terrazoanthus.

- Expand our investigations on zoantharians all along the mainland coast of Ecuador. - Use metabolomics analyses for comparison with other zoantharians in the Pacific and more generally to all oceans in order to contribute to the revision of the systematics of this complex group.

Acknowledgements: SENESCYT is financially supporting the Project of Marine Biodiversity PIC-001, NUI Galway supported the travel of K. Jaramillo. Special thanks also go to CENAIM researchers B. Chalen, C. Dominguez, A. Lavorato, for their precious help in the collection of the specimens, Dr. S. Harii for her help in obtaining the DNA sequences.

References: 1. Ivanišević et al. 2011 Metabolomics 7 (2): 289-304; 2. Cachet et al. 2015 Sci. Rep 5:8282; 3. Reimer & Fujii. 2010 ZooKeys 42:1-36; 4. Reimer & Hickman. 2008 Coral Reefs 27: 641-654; 5. Bo et al. 2012 Pacific Science, vol. 66, no. 1:63-81.