Molecular taxonomy of some sponges (Demospongiae) using ribosomal (18s rrna) and partial mitochondrial (coi) genes

ACADEMIA JOURNAL OF BIOLOGY 2018, 40(4): 67–75<br /> DOI: 10.15625/2615-9023/v40n4.12705<br /> <br /> <br /> <br /> <br /> MOLECULAR TAXONOMY OF SOME SPONGES (Demospongiae) USING<br /> RIBOSOMAL (18S rRNA) AND PARTIAL MITOCHONDRIAL (COI) GENES<br /> <br /> Ton That Huu Dat1*, Nguyen Thi Kim Cuc2, Pham Viet Cuong1<br /> 1<br /> Mien Trung Institute for Scientific Research, VAST<br /> 2<br /> Institute of Marine Biochemistry, VAST<br /> <br /> ABSTRACT<br /> Sponges, the most ancient multicellular metazoan, were widely distributed across habitats.<br /> Vietnam is known to possess a high biodiversity of sponges, however, they are mostly<br /> identified based on morphological characteristics and lack the molecular data. In the current<br /> study, the phylogenetic relationship of some sponges (Demospongiae) in Vietnam was<br /> constructed using two independent markers (COI and 18S rRNA). In this paper the individual<br /> markers (COI and 18S rRNA) were successfully used to identify some sponge taxa at the<br /> species level. The obtained results showed the congruence of molecular taxonomy using two<br /> independent markers. However, our study showed that a combination of the two markers<br /> provided more information and supported better for sponge identification. At order level, the<br /> COI tree and 18S rRNA tree also recovered the same clades, indicating the congruence of COI<br /> and 18S rRNA genes in sponge classification. However, branching order of the clades in COI<br /> tree was weakly supported and slightly different from those in 18S rRNA tree.<br /> Keywords: Demospongiae, 18S rRNA, COI, Phylogenetic tree, Porifera; Mitochondrial genes.<br /> <br /> Citation: Ton That Huu Dat, Nguyen Thi Kim Cuc, Pham Viet Cuong, 2018. Molecular taxonomy of<br /> some sponges (demospongiae) using ribosomal (18S RRNA) and partial mitochondrial (COI) genes.<br /> Academia Journal of Biology, 40(4): 67–75. https://doi.org/10.15625/2615-9023/v40n4.12705.<br /> *Corresponding author email: huudat96@gmail.com<br /> Received 26 June 2018, accepted December 2018<br /> <br /> INTRODUCTION known species was only a half of estimated<br /> Sponges (phylum Porifera) are the most number of species (van Soest et al., 2012).<br /> simple and ancient metazoan. The sponges Initially, the identification of sponges is<br /> were appeared on Earth since at least 650 based mainly on the traditional method using<br /> million years ago (Maloof et al., 2010), and morphological characteristics (e.g., the<br /> widely distributed across geographical and skeletal structure, spicule, and external<br /> bathymetrical habitats (Bell, 2008). At least morphology), in which the skeletal and<br /> 8.500 valid species has been described in specular features are the most frequently used<br /> phylum Porifera belonging to four classes, 25 (Hooper & Soest, 2002). However, the<br /> orders, 128 families, and 680 genera. The paucity and plasticity of morphological<br /> Demospongiae is known as the most characters of sponge result in challenges of<br /> morphologically diverse and one of the richest sponge identification and increase the cryptic<br /> classses of the Porifera (> 85% of total species and homoplasy speciation (van Soest et al.,<br /> number) (van Soest et al., 2012). Until now, 2012). The introduction and development of<br /> sponge classification has been increasingly molecular techniques have significantly<br /> concerned of scientists due to many new contributed to our understanding of<br /> species have not discovered. The number of phylogenetic relationships and evolution of<br /> <br /> <br /> 67<br />  Ton That Huu Dat et al.<br /> <br /> <br /> sponge systematics. Molecular data have still need to be done. In this study, we use two<br /> provided new insights on the identification of different phylogenetic markers (COI and 18S<br /> sponge, particularly in some sponge taxa rRNA) to identify some Vietnamese<br /> which morphological characters are few demosponges and test the congruence of sponge<br /> (Cárdenas et al., 2012). identification based on independent markers.<br /> In Vietnam up to now, at least 299 sponge<br /> MATERIALS AND METHODS<br /> species belonging to 124 genera, 65 families, 18<br /> orders and 4 classes has, of which the Collection of sponge samples<br /> Demospongiae occupied 281 species (94% of Sponge specimens were collected by<br /> the total of detected species) (Quang, 2013). SCUBA diving at Vinh Moc (Quang Tri),<br /> However, most of these sponges were identified Lang Co (Thua Thien Hue) and Hon Mun<br /> based on morphological characteristics. (Nha Trang) (Table 1). Samples were stored<br /> Therefore, the genetic variation and in containers with seawater and kept at (-)<br /> phylogenetic relationship of the sponge species 20°C for molecular analysis.<br /> <br /> Table 1. Sponge samples in our study<br /> Accession number<br /> Code Taxon Date Site Coordinates<br /> 18S rRNA COI<br /> Amphimedon 107°07'01.4"E;<br /> MT1.2015 Jan-2015 Quang Tri KY947243 KY947259<br /> compressa 17°05'08.6"N<br /> Xestospongia 107°07'01.4"E;<br /> MT2.2015 Jan-2015 Quang Tri KY947244 KY947260<br /> testudinaria 17°05'08.6"N<br /> Rhabdastrella 107°07'01.4"E;<br /> MT3.2015 Jan-2015 Quang Tri KY947245 KY947261<br /> globostellata 17°05'08.6"N<br /> Rhabdastrella 107°07'01.4"E;<br /> MT4.2015 Jan-2015 Quang Tri KY947246 KY947262<br /> globostellata 17°05'08.6"N<br /> 107°07'01.4"E;<br /> MT5.2015 Axos cliftoni Jan-2015 Quang Tri KY947247 KY947263<br /> 17°05'08.6"N<br /> 107°07'01.4"E;<br /> MT6.2015 Clathria reinwardti Jan-2015 Quang Tri KY947248 KY947264<br /> 17°05'08.6"N<br /> Amphimedon 108°02'35.9"E;<br /> MT7.2015 Mar-2015 Thua Thien Hue KY947249 KY947265<br /> compressa 16°19'58.5"N<br /> 108°02'35.9"E;<br /> MT8.2015 Clathria reinwardti Mar-2015 Thua Thien Hue KY947250 KY947266<br /> 16°19'58.5"N<br /> Rhabdastrella 108°02'35.9"E;<br /> MT9.2015 Mar-2015 Thua Thien Hue KY947251 KY947267<br /> globostellata 16°19'58.5"N<br /> Amphimedon 108°02'35.9"E;<br /> MT10.2015 Mar-2015 Thua Thien Hue KY947252 KY947268<br /> compressa 16°19'58.5"N<br /> 108°02'35.9"E;<br /> MT11.2015 Clathria reinwardti Mar-2015 Thua Thien Hue KY947253 KY947269<br /> 16°19'58.5"N<br /> 109°15'05.6"E;<br /> MT12.2015 Tedania ignis May-2015 Nha Trang KY947254 KY947270<br /> 12°10'35.4"N<br /> Xestospongia 109°15'05.6"E;<br /> MT13.2015 May-2015 Nha Trang KY947255 KY947271<br /> testudinaria 12°10'35.4"N<br /> 109°15'05.6"E;<br /> MT14.2015 Tedania ignis May-2015 Nha Trang KY947256 KY947272<br /> 12°10'35.4"N<br /> Xestospongia 109°15'05.6"E;<br /> MT15.2015 May-2015 Nha Trang KY947257 KY947273<br /> testudinaria 12°10'35.4"N<br /> Spheciospongia 109°15'05.6"E;<br /> MT16.2015 May-2015 Nha Trang KY947258 -<br /> verparium 12°10'35.4"N<br /> <br /> <br /> <br /> 68<br />  Molecular taxonomy of some sponges (demospongiae)<br /> <br /> <br /> DNA extraction, PCR amplification of 18S MEGA7.0 (Kumar et al., 2016). The<br /> rRNA and COI genes reliability of clades on the phylogenetic tree<br /> Sponge tissue (500mg) was used to extract was assessed based on bootstrap values of<br /> genomic DNA using DNeasy Blood & Tissue 1000 replicates. Sequences were deposited in<br /> Kit (Qiagen, Germany) according to the GenBank under accession numbers:<br /> manufacturer’s protocol. The concentration of KY947243-KY947258 (18S rRNA genes)<br /> extracted DNA was measured by a Nanodrop and KY947259-KY947273 (COI genes).<br /> 1000 spectrophotometer (Nanodrop<br /> RESULTS AND DISCUSSION<br /> Technologies, Wilmington, DE), and its<br /> integrity was examined by gel electrophoresis Molecular taxonomy of sponges based on<br /> on agarose gel 1% (w/v). The extracted DNA 18S rRNA gene<br /> was dissolved in TE buffer and stored at (- The nearly full-length 18S rRNA gene<br /> )20°C for further analysis. fragments of all 16 sponge specimens were<br /> The 18S rRNA genes (~ 1800 bp) and amplified successfully. The BLAST results<br /> partial COI gene fragments (~ 650 bp) were showed that these 18S rRNA genes exhibited<br /> amplified from extracted DNA using the high similarity with other 18S rRNA<br /> primer pairs EukF/EukR (Medlin et al., 1988) sequences on NCBI (96.4−100%) (Table 2).<br /> and jgLCO1490/jgHCO2198 (Geller et al., Results showed topology of phylogenetic<br /> 2013), respectively. The PCR products were trees using different methods was mostly<br /> cloned into the pCRTM2.1 vector (TA Cloning agreement with slight difference in support<br /> Kit, Invitrogen) according to the value. The 18S rRNA phylogenetic tree of<br /> manufacturer’s protocol. Positive clones were collected samples showed phylogenetically<br /> sequenced on DNA Analyzer (ABI PRISM diverse taxa of sponge specimens including 5<br /> orders, 7 families, and 7 genera. Half of the<br /> 3100, Applied Bioscience).<br /> samples were identified to species level based<br /> Construction of phylogenetic tree on their positions on the phylogenetic tree and<br /> Sequences were trimmed to their high similar level (> 99%) to referred<br /> remove low-quality ends using Bioedit version sequences (MT1.2015, MT7.2015, and<br /> 7.2.5. The vector contamination was MT10.2015 belonged to Amphimedon<br /> removed using VecScreen compressa; MT16.2015 belonged to<br /> (https://www.ncbi.nlm.nih.gov/tools/vecscreen/. Spheciospongia vesparium; MT3.2015,<br /> Forward and reverse 18S rRNA sequences MT4.2015, and MT9.2015 belonged to<br /> were assembled to obtain near full-length Rhabdastrella globostellata). Although the<br /> fragments. Sequences in our study and the 18S rRNA sequences of other samples also<br /> had high similar level to those of reference<br /> most their closely related sequences obtained<br /> sequences, they were only identified to genus<br /> from BLAST program (nr/nt) were aligned<br /> level due to their positions on the<br /> using Clustal W and Muscle algorithms on<br /> phylogenetic tree were at the same branch<br /> software MEGA 7.0 (Kumar et al., 2016).<br /> with different species of the same genera (e.g.,<br /> Phylogenetic trees for 18S rRNA and COI MT14.2015 and MT12.2015 in genus<br /> sequences were created using Maximum Tedania, MT6.2015, MT8.2015, and<br /> Likelihood (ML), Neighbor-joining (NJ), and MT11.2015 in genus Clathria). Remaining<br /> Maximum parsimony (MP) with Kimura 2- samples (MT2, MT13, and MT15) were not<br /> parameter model for ML and NJ, and Subtree- identified to species level because of their low<br /> pruning-regrafting (SPR) for MP using similarity (96.4−96.7%) to referred sequences.<br /> <br /> <br /> 69<br />  Ton That Huu Dat et al.<br /> <br /> <br /> Molecular taxonomy of sponges based on on phylogenetic tree of COI genes also given<br /> COI gene similar results to identification based on the<br /> 18S rRNA genes at genus level, except for<br /> The partial COI genes from 15 out of 16 MT5.2015 belonged to Tethyida and<br /> sponge specimens were successfully amplified Axinellida clade. Fourteen out of 15 COI<br /> and sequenced. The PCR product of the genes could be identified to species level<br /> sample MT16.2015 was very weak and was based on their positions on the phylogenetic<br /> failed in sequencing. The COI gene sequences tree and their high similar level (> 99%) to<br /> in our study displayed high similarity with referred sequences. The COI sequence of<br /> other COI gene sequences on NCBI (99 − MT5.2015 could not be identified at a lower<br /> 100%) (Table 2). Constructions of the level (genus or species) because it was<br /> phylogenetic tree using different algorithms positioned at the same branch of two<br /> and methods indicated the topological species belonging to two genera, Axos and<br /> agreement of phylogenetic trees (Fig. 2). Stelligera.<br /> Identification of the sponge specimens based<br /> <br /> Table 2. The similarity of sequences in our study with reference sequences on NCBI<br /> 18S rRNA COI<br /> Code Similarity Similarity<br /> Closely reference sequence Closely reference sequence<br /> (%) (%)<br /> Amphimedon compressa, Amphimedon compressa,<br /> MT1.2015 99.9 99.3<br /> EU702409 EU237474<br /> Xestospongia testudinaria,<br /> MT2.2015 Xestospongia muta, AY621510 96.4 100<br /> HQ452960<br /> Rhabdastrella globostellata, Rhabdastrella globostellata,<br /> MT3.2015 99.9 99.8<br /> KC902160 HM592673<br /> Rhabdastrella globostellata, Rhabdastrella globostellata,<br /> MT4.2015 99.9 99.8<br /> KC902160 HM592673<br /> MT5.2015 Axos cliftoni, EF654523 99.2 Axos cliftoni, AY561974 99.4<br /> MT6.2015 Clathria reinwardti, KC902087 99.9 Clathria reinwardti, HE611598 100<br /> Amphimedon compressa, Amphimedon compressa,<br /> MT7.2015 99.8 98.7<br /> EU702409 EU237474<br /> MT8.2015 Clathria reinwardti, KC902087 99.9 Clathria reinwardti, HE611598 99.8<br /> Rhabdastrella globostellata, Rhabdastrella globostellata,<br /> MT9.2015 100 99.8<br /> KC902160 HM592673<br /> MT10.201 Amphimedon compressa, Amphimedon compressa,<br /> 99.8 99.0<br /> 5 EU702409 EU237474<br /> MT11.201<br /> Clathria reinwardti, KC902087 99.9 Clathria reinwardti, HE611598 99.8<br /> 5<br /> MT12.201<br /> Tedania ignis, AY737642 99.2 Tedania ignis, DQ133896 99.8<br /> 5<br /> MT13.201 Xestospongia testudinaria,<br /> Xestospongia muta, AY621510 96.7 100<br /> 5 HQ452960<br /> MT14.201<br /> Tedania ignis, AY737642 99.4 Tedania ignis, DQ133896 99.8<br /> 5<br /> MT15.201 Xestospongia testudinaria,<br /> Xestospongia muta, AY621510 96.5 100<br /> 5 HQ452960<br /> MT16.201 Spheciospongia vesparium,<br /> 99.9 - -<br /> 5 AY734440<br /> <br /> <br /> <br /> <br /> 70<br />  Molecular taxonomy of some sponges (demospongiae)<br /> <br /> <br /> The congruence of sponge classification (Duran et al., 2004; Redmond et al., 2007;<br /> based on 18S rRNA and COI gene Sipkema et al., 2003). For example, the 18S<br /> rRNA sequences of two species belonging to<br /> Phylogenetic trees of 18S rRNA and COI<br /> two different genera (Amphimedon<br /> genes indicated agreement of sponge<br /> quenslandica and Haliclona (?gellius) sp.) are<br /> taxonomy. At the genus level, the same<br /> nearly identical (Sipkema et al., 2009) and<br /> genera were recovered based on two<br /> unable to identification of these species using<br /> independent marker genes (18S and COI),<br /> the 18S rRNA gene. Similarly, the COI<br /> except for specimen MT5.2015. Six out of 16<br /> sequences are often too conserved in sponges<br /> specimens showed also the agreement of<br /> to resolve population-level relationships<br /> taxonomy at the species level (MT1.2015,<br /> (Duran et al., 2004). However, the individual<br /> MT7.2015, MT10.2015 belonged to<br /> sequences display different evolution rates of<br /> Amphimedon compressa, and MT4.2015,<br /> sponges (Wang & Lavrov, 2008) and may be<br /> MT3.2015, MT9.2015 belonged to<br /> more or less suitable for a specific<br /> Rhabdastrella globostellata). Some specimens<br /> classification. The mitochondrial sequences<br /> could not be identified at species level using<br /> appear to evolve higher in some sponge taxa<br /> individual marker; however, using a<br /> and have been used effectively for studies<br /> combination of both markers allowed to<br /> population level (Dailianis et al., 2011;<br /> identify them at the species level. Specimens<br /> Escobar et al., 2012) and detection of cryptic<br /> MT12.2015 and MT14.2015 (Tedania), and<br /> species (Andreakis et al., 2012; de Paula et al.,<br /> MT6.2015, MT8.2015, and MT11.2015<br /> 2012). These findings revealed that the<br /> (Clathria) could not be identified to species<br /> individual markers often provide different<br /> level because the variation of 18S rRNA<br /> rates of sponge evolution, combination of<br /> sequences between species in two genera<br /> different phylogenetic markers, therefore, are<br /> Tedania and Clathria was very low. However,<br /> expected to be more informative of sponge<br /> the COI phylogenetic tree of these specimens<br /> phylogeny at different levels.<br /> showed better resolution, and they could be<br /> identified to species level. In contrast, At a higher classification level (orders),<br /> specimen MT5.2015 could not be identified to phylogenetic trees of 18S rRNA and COI<br /> species level using only COI gene, however, sequences recovered the same clades;<br /> this specimen could be identified to species however, the branching order of clades in two<br /> level (Axos cliftoni) using 18S rRNA genes. In phylogenetic trees was different. In addition,<br /> the case of specimens MT2.2015, support value of nodes in 18S rRNA<br /> MT13.2015, and MT15.2015, the 18S rRNA phylogenetic tree was better than those in COI<br /> sequences showed low similarity with phylogenetic tree (Figs. 1 & 2). The<br /> reference sequence on NCBI (< 98%), and previously studies reveal that phylogenetic<br /> could not be identified to species level relationship of some orders (e.g.,<br /> because the 18S rRNA sequence of Haplosclerida) are different using 18S, 28S<br /> Xestospongia testudinaria was not available rRNA, and COI sequences (McCormack et<br /> from NCBI. However, the COI sequences of al., 2002; Nichols, 2005). Morrow et al.<br /> these specimens showed 100% similarity with (2012) also investigated the congruence of<br /> nuclear genes (18S and 28S rRNA) and<br /> the COI sequence of Xestospongia<br /> mitochondrial gene (COI) in sponges<br /> testudinaria and could be identified as<br /> (Demospongiae), and showed that the<br /> Xestospongia testudinaria. phylogenetic tree based on COI sequences<br /> The findings in our study are consistent recovered the same clades and same genera as<br /> with previous studies. The previous studies the 18S and 28S rRNA tree. However, the<br /> have suggested that low variation and slow branching order in the COI tree is different<br /> evolution rate of 18S rRNA and COI genes and less resolution than in 18S and 28S rRNA<br /> may result in difficulty in identification of trees (Morrow et al., 2012). The difference<br /> some sponges at the lower taxonomic level may result from different evolution rate of<br /> <br /> <br /> 71<br />  Ton That Huu Dat et al.<br /> <br /> <br /> 18S rRNA and COI genes. For example, the Verticillitida. In contrast, the mitochondrial<br /> evolution rate of mitochondrial sequences is sequences display lower evolution rate<br /> higher than those of rRNA sequences for than rRNA sequences for the order<br /> some orders such as Dictyoceratida and Homoclerophorida (Lavrov et al., 2008).<br /> <br /> <br /> <br /> <br /> Figure 1. Phylogeny of 18S rRNA gene sequences of sponges in our study (blue bold letters)<br /> and from NCBI. The tree topology was obtained from NJ. Individual bootstrap values from ML,<br /> NJ, MP with alignment methods ClustalW (Clu) and Muscle (Mus) are located in the upper-<br /> right box and correspond to circled numbers on tree nodes. Solid lines indicate well-supported<br /> branches (support values greater than 50% for all criteria) and dashed lines indicate weakly<br /> supported branches<br /> <br /> <br /> 72<br />  Molecular taxonomy of some sponges (demospongiae)<br /> <br /> <br /> <br /> <br /> Figure 2. Phylogeny of partial COI gene sequences of sponges in our study (blue bold letters)<br /> and from NCBI. The tree topology was obtained from neighbor-joining (NJ) analysis. Individual<br /> bootstrap values from maximum likelihood (ML), NJ, maximum parsimony (MP) with<br /> alignment methods ClustalW (Clu) and Muscle (Mus) are located in the lower-right box and<br /> correspond to circled numbers on tree nodes. Solid lines indicate well-supported branches<br /> (support values greater than 50% for all criteria) and dashed lines indicate weakly supported<br /> branches.<br /> <br /> CONCLUSION taxonomy using two independent markers,<br /> Based on two molecular markers (18S however order of the clades in COI tree was<br /> rRNA and COI), sponges (Demospongiae) in different and less supported than from those in<br /> Vietnam were identified. The obtained results 18S rRNA. Combination of the two markers<br /> showed the congruence of molecular supported better for sponge identification.<br /> <br /> <br /> 73<br />  Ton That Huu Dat et al.<br /> <br /> <br /> Acknowledgements: This work was and Atlantic range of the sponge Crambe<br /> supported by Vietnamese Ministry of crambe (Poecilosclerida). Mar. Biol.,<br /> Science and Technology under grant 144(1): 31−35.<br /> number: ĐTĐLCN.17/14." Escobar D., Zea S., Sánchez J. 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