Phenotypic Variability in the Caribbean Orange Icing Sponge Mycale Laevis (Demospongiae: Poecilosclerida)
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Hydrobiologia DOI 10.1007/s10750-011-0806-1 SPONGE RESEARCH DEVELOPMENTS Phenotypic variability in the Caribbean Orange Icing sponge Mycale laevis (Demospongiae: Poecilosclerida) Tse-Lynn Loh • Susanna Lo´pez-Legentil • Bongkeun Song • Joseph R. Pawlik Received: 22 February 2011 / Accepted: 18 June 2011 Ó Springer Science+Business Media B.V. 2011 Abstract Sponge species may present several mor- Aegogropila is polyphyletic and the subgenus Mycale photypes, but sponges that are morphologically similar is paraphyletic. All 4 morphotypes formed a mono- can be separate species. We investigated morpholog- phyletic group within Mycale, and no genetic differ- ical variation in Mycale laevis, a common Caribbean ences were observed among them. Spicule lengths did reef sponge. Four morphotypes of M. laevis have been not differ among the 4 morphotypes, but the dominant observed (1) orange, semi-cryptic, (2) orange, massive, megasclere in samples collected from Florida and the (3) white, semi-cryptic, and (4) white, massive. Bahamas was the strongyle, while those from Panama Samples of M. laevis were collected from Key Largo, had subtylostyles. Our data suggest that the 4 morpho- Florida, the Bahamas Islands, and Bocas del Toro, types constitute a single species, but further studies Panama. Fragments of the 18S and 28S rRNA would be necessary to determine whether skeletal ribosomal genes were sequenced and subjected to variability is due to phentotypic or genotypic plasticity. phylogentic analyses together with sequences obtained for 11 other Mycale species and additional sequences Keywords Porifera 18S rRNA 28S rRNA retrieved from GenBank. Phylogenetic analyses con- Ribosomal DNA CaribbeanÁ MorphotypeÁ Á Á Á Á firmed that the genus Mycale is monophyletic within Genotype the Order Poecilosclerida, although the subgenus Introduction Ecological studies conducted in areas of high biodi- versity may sacrifice precision in lower-level taxon Guest editors: M. Maldonado, X. Turon, M. A. Becerro & M. J. Uriz / Ancient animals, new challenges: developments sampling in order to gain a broader understanding of in sponge research large-scale ecosystem processes. In community sur- veys, marine invertebrates are usually not completely T.-L. Loh (&) B. Song J. R. Pawlik identified to the species-level due to difficulties in Center for MarineÁ Science,Á University of North Carolina Wilmington, 5600 Marvin K Moss Lane, Wilmington, field identification or lack of resolution in available NC 28409, USA taxonomic keys (e.g., Chou et al., 2004; Micheli e-mail: [email protected] et al., 2005; Chapman et al., 2010). Sponges (Phylum Porifera) are a good example of a taxon sampled at S. Lo´pez-Legentil Department of Animal Biology (Invertebrates), University low resolution in many community surveys. In the of Barcelona, 643, Diagonal Ave, Barcelona 08028, Spain widely used benthic survey methodologies employed 123 Hydrobiologia by the Global Coral Reef Monitoring Network the field may be separate species (Klautau et al., (GCRMN, http://www.gcrmn.org) and Reef Check 1999; Miller et al., 2001; Duran & Ru¨tzler, 2006; (http://www.reefcheck.org), coral reef sponges are Wulff, 2006a; Blanquer & Uriz, 2007; Blanquer classified into a single group (Wilkinson, 2008). et al., 2008). However, this single phylum is comprised of *7000 The Orange Icing Sponge Mycale laevis (Carter species (Hooper & Van Soest, 2002). Furthermore, 1882, Order Poecilosclerida, subgenus Mycale) ranks sponges are important members of the aquatic eco- as one of the 10 most common sponges on Caribbean systems they inhabit, both in terms of abundance and coral reefs (Pawlik et al., 1995), and has been ecosystem function, they compete for space with reported as a common associate of scleractinian other benthic organisms, they are dominant suspen- corals (Goreau & Hartman, 1966; Hill, 1998). As is sion feeders, and they provide habitat for a large and often observed for other sponge species, M. laevis has diverse number of other invertebrates (Corredor et al., more than one growth form. It has been described as 1988; Pile et al., 1996; Diaz & Rutzler, 2001; Henkel both semi-cryptic, a thinly encrusting form with & Pawlik, 2005; Southwell et al., 2008). Given their most of the biomass growing under coral or other ecological importance, why are sponges seldom hard substrata (Fig. 1a; Wulff, 1997), and massive, a identified to the level of species in the field? fleshy, apparent form that grows on the upper surface The process of identifying sponge species can be of substrata (Fig. 1b; Randall & Hartman, 1968; difficult. Sponge identification is based on morpho- Wulff, 2006b). On reefs off Bocas del Toro, Panama logical attributes such as color, size and shape, the and the Bahamas Islands, a white morph of M. laevis presence, and arrangement of the spiculate and has also been described (Fig. 1c, d; Collin et al., fibrous skeleton, and most particularly, spicule type 2005). This white morphotype is more common than and size. This presents a challenge as sponges are the orange on some reefs at Bocas del Toro, exhibits morphologically plastic, often have different color both encrusting and fleshy forms (observed at the morphs, and can change in shape and size due to Bahamas Islands and Panama, respectively), and environmental conditions (Palumbi, 1986) or biotic frequently grows in association with corals in the factors such as predation (Loh & Pawlik, 2009). same manner as the orange morphotype (Loh per- Spicule morphology, by far the most important sonal observation). The white morphotype is super- character state, can also vary among different habitats ficially similar to the orange, having a compressible and growing conditions (Hooper, 1985; McDonald texture, a rough external surface, and osculae ringed et al., 2002). Furthermore, sponges can incorporate by thin membranous collars with vertical white lines abiotic materials from the environment or spicules (Collin et al., 2005). from other sponges into their skeletons (Sollas, 1908; In this study, we investigated genetic differences Teragawa, 1986; Hooper & Van Soest, 2002). among 4 morphotypes of M. laevis using partial 18S Conversely, sponges that look almost identical in and 28S rRNA ribosomal gene sequences. Samples Fig. 1 The four morphotypes of Mycale laevis: a orange, semi-cryptic, b orange, massive, c white, massive, d white semi-cryptic 123 Hydrobiologia were collected from the coral reefs off Key Largo, collected between 2007 and 2010 and immediately Florida, Bocas del Toro, Panama, and the Bahamas preserved in 95–100% ethanol, frozen at -20°C, or Islands. In addition, we sequenced 11 species of freeze-dried. All samples were then stored at -20°C Mycale, and retrieved 5 other sequences from Gen- until they were extracted. bank to perform phylogenetic analyses and determine Caribbean species were identified by comparing the taxonomic status of the 4 morphotypes of morphological and spicule characters with Hajdu & M. laevis. We also examined spicule morphology Rutzler (1998) and with the sponge voucher collec- and diversity and calculated spicule dimensions to tion at the Bocas Research Station of the Smithsonian compare among the 4 morphotypes of M. laevis. Tropical Research Institute (specimens submitted by C. Diaz and R. Thacker). Other Mycale species were identified by their respective collectors. Materials and methods DNA extraction and sequencing Sample collection DNA was extracted using the Puregene kit (Gentra When possible, three samples of each morphotype of Systems). The most commonly genetic markers used M. laevis were collected from each of the study sites to investigate taxonomic and phylogenetic issues in where they occurred (Table 1). The orange semi- sponges are the 18S and 28s rRNA genes, and the cryptic morphotype was present at all the sites, while mitochondrial gene cytochrome oxidase I (COI). the orange massive morphotype was found only at Here, we designed the primer set 18sMycale01F Bocas del Toro. The white semi-cryptic morphotype 50-ATAACTGCTCGAACCGTATGGCCT-30 and was collected at Tuna Alley reef, Bahamas (2 18SMycale01R 50-AAACGCTAACATCCACCGAT samples), while the white massive morphotype was CCCT-30 based on an 18S rRNA sequence of found only at Bocas del Toro. For our 3 sampling M. fibrexilis available from GenBank (AF100946) locations, the massive morphotypes (both orange and to amplify a fragment of 786 bp from the 18S rRNA white), were only found at Bocas del Toro. gene. However, no amplification could be obtained One to three samples of the following 11 Mycale for the species M. lingua, M. parishi, Mycale cf. species were collected and added to the analysis of lingua, Mycale sp. J57, 1 sample of the orange samples of M. laevis to enhance phylogenetic tree massive morphotype, and all white morphotypes of resolution: M. lingua (Bowerbank 1866, collected in M. laevis, with the 18S rRNA primers described Norway by P. Cardenas) and M. grandis (Gray 1867, above. Amplification was finally obtained with the Singapore, S. C. Lim) from the subgenus Mycale; primer set 18sMycale02F 50-CAACGGGTGACG M. sulevoidea (Sollas 1902, Singapore, S. C. Lim), GAGAATTA-30 and 18sMycale02R 50-TTTCAG M. adhaerens (Lambe 1893, Hong Kong, Y. H. Wong CCTTGCGACCATACTC-30, which was designed and P. Y. Qian), and M. carmigropila (Hajdu & based on the consensus sequence of the poecilosclerid Rutzler, 1998, Panama, T. L. Loh) from the subgenus library available at GenBank. Amplification of a Aegogropila; M. parishi (Bowerbank 1875, Singa- fragment from the 28S rRNA gene