Plakortis2015_ESM 3
Molecular differences between Plakortis angulospiculatus and P. halichondrioides.
Maximum likelihood phylogeny of Homoscleromorpha based on DNA sequencing of a 400 bp
fragment at the 5’ end of the gene encoding the large (28S) subunit of nuclear ribosomal RNA.
To confirm species identifications for this study, genomic DNA was extracted from specimens
identified as Plakortis angulospiculatus and P. halichondrioides, using the Wizard Genomic
DNA Purification Kit (Promega), following the manufacturer’s protocol for animal tissue. PCR
amplification and sequencing followed the methods described by Thacker and Starnes (2003).
Sequences obtained (labeled in bold font) were deposited in GenBank under accession numbers
KP698747-KP698749. Sequences were aligned to other 28S sequences from representatives of
Haploscleromorpha deposited in GenBank using the MAFFT alignment algorithm with its
default parameters (Katoh and Standley 2013). This maximum likelihood phylogeny was
constructed using RAxML software (Stamatakis et al. 2008). Scale bar indicates 0.2 substitutions
per site. Numbers at nodes indicate percentage bootstrap support; values less than 50% are not
shown. These sequences clearly distinguished between P. angulospiculatus and P.
halichondrioides. Although this short fragment of DNA can be used to identify species, it may
not accurately reconstruct relationships among species (Thacker et al. 2013).
HM118553 Corticium candelabrum
100
HM118555 Corticium sp. 2 EG-2011
100
HM118554 Corticium sp. 1 EG-2011
100
HM118559 Plakina crypta
HM118560 Plakina trilopha
100
HM118558 Plakina jani
60
BA400-300-536 Plakortis angulospiculatus
KP081745 Plakortis sp. 2 JV-2014
74
100 KP081744 Plakortis sp. 2 JV-2014
56
86
KP081746 Plakortis sp. 2 JV-2014
HM118563 Plakinastrella sp. EG-2011
100
100
HM118562 Plakinastrella onkodes
HM118556 Plakortis simplex
98
HM118557 Plakortis halichondrioides
100
KC869492 Plakortis halichondrioides
KP081742 Plakortis halichondrioides
91
BA400-25-541 Plakortis halichondrioides
BA400-70-539 Plakortis halichondrioides
KP081741 Plakortis halichondrioides
JX462770 Oscarella nicolae
100
JX462781 Oscarella kamchatkensis
57
HM118552 Pseudocorticium jarrei
100
100
HM118550 Oscarella malakhovi
EF654519 Oscarella carmela
100
95
JX462780 Oscarella tuberculata
JX462774 Oscarella lobularis
0.2
Supplemental References
Katoh K, Standley DM (2013) MAFFT multiple sequence alignment software version 7:
improvements in performance and usability. Mol Biol Evol 30:772-780
Stamatakis A, Hoover P, Rougemont J (2008) A rapid bootstrap algorithm for the RAxML webservers. Systematic Biol 75:758-771
Thacker RW, Hill AL, Hill MS, Redmond NE, Collins AG, Morrow CC, Spicer L, Carmack CA,
Zappe ME, Pohlmann D, Hall C, Diaz MC, Bangalore PV (2013) Nearly complete 28S rRNA
gene sequences confirm new hypotheses of sponge evolution. Integrative Comp Biol 53:373-387
Thacker RW, Starnes S (2003) Host specificity of the symbiotic cyanobacterium Oscillatoria
spongeliae in marine sponges, Dysidea spp. Mar Biol 142:643-648