Parendozoicomonas callyspongiae sp. nov. Isolated from a Marine Sponge, Callyspongia elongate, and Reclassification of Sansalvadorimonas verongulae as Parendozoicomonas verongulae comb. nov.

A strictly aerobic Gram-negative bacterium, designated 2012CJ34-2T, was isolated from marine sponge to Chuja-do in Jeju-island, Republic of Korea and taxonomically characterized. Cells were catalase- and oxidase-positive, and non-motile rods (without flagella). Growth was observed at 15-42 °C (optimum, 30 °C), pH 6-9 (optimum, pH 7), and in the presence of 0.5-10% (w/v) NaCl (optimum, 2-3%). The major cellular fatty acid and respiratory quinones were identified summed feature 3 (C16:1 ω7c/C16:1 ω6c), and Q-8 and Q-9, respectively. The polar lipids comprised diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, an unidentified aminophospholipid, two unidentified phospholipids, and three unidentified lipids. The DNA G+C content was 48.0 mol%. Phylogenetic analyses based on 16S rRNA gene and whole genome sequences showed that strain 2012CJ34-2T formed a clade with Parendozoicomonas haliclonae S-B4-1UT and Sansalvadorimonas verongulae LMG 29871T within the family Endozoicomodaceae. Genome relatedness values, including dDDH, ANI and AF, and AAI and POCP, among strain 2012CJ34-2T, P. haliclonae S-B4-1UT, and S. verongulae LMG 29871T were within the range of the bacterial genus cut-off values. Based on the phylogenetic, chemotaxonomic, and genomic analyses, strain 2012CJ34-2T represents a novel bacterial species of the family Endozoicomodaceae, for which the name Parendozoicomonas callyspongiae sp. nov. is proposed. The type strain is 2012CJ34-2T (= KACC 22641T = LMG 32581T). Additionally, we proposed the reclassification of Sansalvadorimonas verongulae of the family Hahellaceae as Parendozoicomonas verongulae of the family Endozoicomonadaceae.

Targeting antimalarial metabolites from the actinomycetes associated with the Red Sea sponge Callyspongia siphonella using a metabolomic method.

Malaria is a persistent illness that is still a public health issue. On the other hand, marine organisms are considered a rich source of anti­infective drugs and other medically significant compounds. Herein, we reported the isolation of the actinomycete associated with the Red Sea sponge Callyspongia siphonella. Using "one strain many compounds" (OSMAC) approach, a suitable strain was identified and then sub-cultured in three different media (M1, ISP2 and OLIGO). The extracts were evaluated for their in-vitro antimalarial activity against Plasmodium falciparum strain and subsequently analyzed by Liquid chromatography coupled with high-resolution mass spectrometry (LC-HR-MS). In addition, MetaboAnalyst 5.0 was used to statistically analyze the LC-MS data. Finally, Molecular docking was carried out for the dereplicated metabolites against lysyl-tRNA synthetase (PfKRS1). The phylogenetic study of the 16S rRNA sequence of the actinomycete isolate revealed its affiliation to Streptomyces genus. Antimalarial screening revealed that ISP2 media is the most active against Plasmodium falciparum strain. Based on LC-HR-MS based metabolomics and multivariate analyses, the static cultures of the media, ISP2 (ISP2-S) and M1 (M1-S), are the optimal media for metabolites production. OPLS-DA suggested that quinone derivatives are abundant in the extracts with the highest antimalarial activity. Fifteen compounds were identified where eight of these metabolites were correlated to the observed antimalarial activity of the active extracts. According to molecular docking experiments, saframycin Y3 and juglomycin E showed the greatest binding energy scores (-6.2 and -5.13) to lysyl-tRNA synthetase (PfKRS1), respectively. Using metabolomics and molecular docking investigation, the quinones, saframycin Y3 (5) and juglomycin E (1) were identified as promising antimalarial therapeutic candidates. Our approach can be used as a first evaluation stage in natural product drug development, facilitating the separation of chosen metabolites, particularly biologically active ones.

Ruegeria spongiae sp. nov., isolated from Callyspongia elongata.

A Gram-stain-negative, rod-shaped, polar flagellated, aerobic, light-yellow bacterium, designated as 2012CJ41-6T, was isolated from a sponge sample of Callyspongia elongata from Chuja-myeon, Jeju-si, Jeju-do, Republic of Korea. On the basis of 16S rRNA gene sequencing, strain 2012CJ41-6T clustered with species of the genus Ruegeria and appeared closely related to R. halocynthiae DSM 27839T (96.46 % similarity), R. denitrificans CECT 4357T (96.32 %), R. profundi ZGT108T (96.32 %), R. litorea CECT 7639T (96.32 %) and R. atlantica CECT 4292T (96.16 %). The average nucleotide identity and digital DNA-DNA hybridization between strain 2012CJ41-6T and the most closely related strain was 75.3 % and 19.6 %, indicating that 2012CJ41-6T represents a novel species of the genus Ruegeria. Growth occurred at 15-37 °C on marine medium in the presence of 0.5-10 % (w/v) NaCl and at pH 5.5-8.5. The DNA G+C content of the genomic DNA was 60.80 mol%, and ubiquinone-10 (Q-10) was the major respiratory quinone. The major cellular fatty acids (>5 %) were C18 : 1 ω7c and/or C18:1 ω6c (summed feature 8). The polar lipids consisted of phosphatidylglycerol, diphosphatidylglycerol, phosphatidylcholine, one unidentified phospholipid, one unidentified aminolipid, one unidentified aminophospholipid and five unidentified lipids. Physiological and biochemical characteristics indicated that strain 2012CJ41-6T represents a novel species of the genus Ruegeria, for which the name Ruegeria spongiae sp. nov. is proposed. The type strain is 2012CJ41-6T (=KACC 22645T=LMG 32585T).

Muricauda spongiicola sp. nov., isolated from the sponge Callyspongia elongata.

A novel Gram-stain-negative, thin-rod-shaped, aerobic, non-motile and yellow-pigmented marine bacterium (designated strain 2012CJ35-5T) was isolated from a marine sponge Callyspongia elongata sampled in Jeju-do, Republic of Korea. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain 2012CJ35-5T belonged to the family Flavobacteriaceae, and was most closely related to Muricauda olearia KCCM 90075T (96.3 %), Muricauda alvinocaridis JCM 33425T (95.7 %), Muricauda flava KCTC 22665T (95.6 %), Muricauda koreensis KCTC 52351T (95.6 %) and Muricauda eckloniae KCTC 22266T (94.9 %). Average nucleotide identity, amino acid identities and digital DNA-DNA hybridization between strain 2012CJ35-5T and the closest related strain were 72.6, 73.6 and 17.3 % respectively, indicating that strain 2012CJ35-5T represents a novel species of the genus Muricauda. The genome size of strain 2012CJ35-5T is 3.8 Mbp and the G+C content is 43.9 mol%. Strain 2012CJ35-5T contained menaquinone 6 as the major respiratory quinone and phosphatidylethanolamine, four unidentified aminophospholipids and five unidentified lipids as major polar lipids. The fatty acids were mainly (>15 %) defined as C15 : 0 iso (31.7 %), C15 : 1 iso G (29.2 %) and C17 : 0 iso 3OH (18.0 %). Strain 2012CJ35-5T could be distinguished from the other members of the genus Muricauda by a number of chemotaxonomic and phenotypic characteristics. Based on the results of polyphasic taxonomic analysis, strain 2012CJ35-5T (=KACC 22643T=LMG 32584T) represents a novel species within the genus Muricauda, for which the name Muricauda spongiicola sp. nov. is proposed.

Computational Metabolomics Tools Reveal Subarmigerides, Unprecedented Linear Peptides from the Marine Sponge Holobiont Callyspongia subarmigera.

A detailed examination of a unique molecular family, restricted to the Callyspongia genus, in a molecular network obtained from an in-house Haplosclerida marine sponge collection (including Haliclona, Callyspongia, Xestospongia, and Petrosia species) led to the discovery of subarmigerides, a series of rare linear peptides from Callyspongia subarmigera, a genus mainly known for polyacetylenes and lipids. The structure of the sole isolated peptide, subarmigeride A (1) was elucidated through extensive 1D and 2D NMR spectroscopy, HRMS/MS, and Marfey's method to assign its absolute configuration. The putative structures of seven additional linear peptides were proposed by an analysis of their respective MS/MS spectra and a comparison of their fragmentation patterns with the heptapeptide 1. Surprisingly, several structurally related analogues of subarmigeride A (1) occurred in one distinct cluster from the molecular network of the cyanobacteria strains of the Guadeloupe mangroves, suggesting that the true producer of this peptide family might be the microbial sponge-associated community, i.e., the sponge-associated cyanobacteria.

Metabolities from Marine Sponges of the Genus Callyspongia: Occurrence, Biological Activity, and NMR Data.

The genus Callyspongia (Callyspongiidae) encompasses a group of demosponges including 261 described species, of which approximately 180 have been accepted after taxonomic reviews. The marine organisms of Callyspongia are distributed in tropical ecosystems, especially in the central and western Pacific, but also in the regions of the Indian, the West Atlantic, and the East Pacific Oceans. The reason for the interest in the genus Callyspongia is related to its potential production of bioactive compounds. In this review, we group the chemical information about the metabolites isolated from the genus Callyspongia, as well as studies of the biological activity of these compounds. Through NMR data, 212 metabolites were identified from genus Callyspongia (15 species and Callyspongia sp.), belonging to classes such as polyacetylenes, terpenoids, steroids, alkaloids, polyketides, simple phenols, phenylpropanoids, nucleosides, cyclic peptides, and cyclic depsipeptides. A total of 109 molecules have been reported with bioactive activity, mainly cytotoxic and antimicrobial (antibacterial and antifungal) action. Thus, we conclude that polyacetylenes, terpenoids and steroids correspond to the largest classes of compounds of the genus, and that future research involving the anticancer action of the species' bioactive metabolites may become relevant.

Reactive oxygen species generation and mitochondrial dysfunction for the initiation of apoptotic cell death in human hepatocellular carcinoma HepG2 cells by a cyclic dipeptide Cyclo(-Pro-Tyr).

Cyclic dipeptides are increasingly gaining importance as considering its significant biological and pharmacological activities. This study was aimed to investigate the anticancer activity of a dipeptide Cyclo(-Pro-Tyr) (DP) identified from marine sponge Callyspongia fistularis symbiont Bacillus pumilus AMK1 and the underlying apoptotic mechanisms in the liver cancer HepG2 cell lines. MTT assay was done to demonstrate the cytotoxic effect of DP in HepG2 cells and mouse Fibroblast McCoy cells. Initially, apoptosis inducing activity of DP was identified using propidium iodide (PI) and acridine orange/ethidium bromide (AO/EB) dual staining, then it was confirmed by DNA fragmentation assay and western blotting analysis of apoptosis related markers Bax, Bcl-2, cytochrome c, caspase-3 and cleaved poly (ADP-ribose) polymerase (PARP). Rhodamine 123 staining was performed to observe DP effects on the mitochondrial membrane potential (MMP) and DCFH-DA (Dichloro-dihydro-fluorescein diacetate) staining was done to measure the intracellular reactive oxygen species (ROS) levels. The MTT results revealed that DP initiated dose-dependent cytotoxicity in HepG2 cells, but no significant toxicity in mouse Fibroblast McCoy cells treated with DP at the specified concentrations. DP induced apoptosis, which is confirmed by the appearance of apoptotic bodies with PI and AO/EB dual staining, and DNA fragmentation. DP significantly elevated the Bax/Bcl-2 ratio, disrupted the mitochondrial membrane potential (MMP), enhanced cytochrome c release from mitochondria, increased caspase-3 activation, the cleavage of PARP and increased intracellular reactive oxygen species (ROS) levels. Besides this, DP successfully inhibited the phosphorylation of PI3K, AKT and increased PTEN expression. These results suggested DP might have anti-cancer effect by initiating apoptosis through mitochondrial dysfunction and downregulating PI3K/Akt signaling pathway in HepG2 cells with no toxicity effect on normal fibroblast cells. Therefore, DP may be developed as a potential alternative therapeutic agent for treating hepatocellular carcinoma.

Antimicrobial Chlorinated 3-Phenylpropanoic Acid Derivatives from the Red Sea Marine Actinomycete Streptomyces coelicolor LY001.

The actinomycete strain Streptomyces coelicolor LY001 was purified from the sponge Callyspongia siphonella. Fractionation of the antimicrobial extract of the culture of the actinomycete afforded three new natural chlorinated derivatives of 3-phenylpropanoic acid, 3-(3,5-dichloro-4-hydroxyphenyl)propanoic acid (1), 3-(3,5-dichloro-4-hydroxyphenyl)propanoic acid methyl ester (2), and 3-(3-chloro-4-hydroxyphenyl)propanoic acid (3), together with 3-phenylpropanoic acid (4), E-cinnamic acid (5), and the diketopiperazine alkaloids cyclo(l-Phe-trans-4-OH-l-Pro) (6) and cyclo(l-Phe-cis-4-OH-d-Pro) (7) were isolated. Interpretation of nuclear magnetic resonance (NMR) and high-resolution electrospray ionization mass spectrometry (HRESIMS) data of 1-7 supported their assignments. Compounds 1-3 are first candidates of the natural chlorinated phenylpropanoic acid derivatives. The production of the chlorinated derivatives of 3-phenylpropionic acid (1-3) by S. coelicolor provides insight into the biosynthetic capabilities of the marine-derived actinomycetes. Compounds 1-3 demonstrated significant and selective activities towards Escherichia. coli and Staphylococcus aureus, while Candida albicans displayed more sensitivity towards compounds 6 and 7, suggesting a selectivity effect of these compounds against C. albicans.

Bioactive Brominated Oxindole Alkaloids from the Red Sea Sponge Callyspongia siphonella.

In the present study, LC-HRESIMS-assisted dereplication along with bioactivity-guided isolation led to targeting two brominated oxindole alkaloids (compounds 1 and 2) which probably play a key role in the previously reported antibacterial, antibiofilm, and cytotoxicity of Callyspongia siphonella crude extracts. Both metabolites showed potent antibacterial activity against Gram-positive bacteria, Staphylococcus aureus (minimum inhibitory concentration (MIC) = 8 and 4 µg/mL) and Bacillus subtilis (MIC = 16 and 4 µg/mL), respectively. Furthermore, they displayed moderate biofilm inhibitory activity in Pseudomonas aeruginosa (49.32% and 41.76% inhibition, respectively), and moderate in vitro antitrypanosomal activity (13.47 and 10.27 µM, respectively). In addition, they revealed a strong cytotoxic effect toward different human cancer cell lines, supposedly through induction of necrosis. This study sheds light on the possible role of these metabolites (compounds 1 and 2) in keeping fouling organisms away from the sponge outer surface, and the possible applications of these defensive molecules in the development of new anti-infective agents.

Callyspongiamides A and B, sterol O-acyltransferase inhibitors, from the Indonesian marine sponge Callyspongia sp.

Callyspongiamides A (1) and B (2), two new sterol O-acyltransferase (SOAT) inhibitors, were isolated from the Indonesian marine sponge Callyspongia sp. together with a known congener, dysamide A (3). The structures of 1 and 2 were elucidated to be polychlorine-containing modified dipeptides based on their spectroscopic data. Compounds 1-3 inhibited both of the SOAT isozymes, SOAT1 and SOAT2, in cell-based and enzyme-based assays.

Callyspongidic Acids: Amphiphilic Diacids from the Tropical Eastern Pacific Sponge Callyspongia cf. californica.

The first chemical study of the marine sponge Callyspongia cf. californica widely distributed along the coasts of the Tropical Eastern Pacific led to the identification of a new family of amphiphilic derivatives called callyspongidic acids. The four isolated metabolites 1-4 feature a hydrophilic diacid end opposed to both an aromatic moiety and a long alkyl chain. They were evaluated against a panel of pathogenic microbes and seven tumoral cell lines, displaying moderate inhibitory properties against the A2058 melanoma cell line with an IC50 of 3.2 µM for callyspongidic acid C13:0 (2).

New Cytotoxic Cyclic Peptide from the Marine Sponge-Associated Nocardiopsis sp. UR67.

A new cyclic hexapeptide, nocardiotide A (1), together with three known compounds-tryptophan (2), kynurenic acid (3), and 4-amino-3-methoxy benzoic acid (4)-were isolated and identified from the broth culture of Nocardiopsis sp. UR67 strain associated with the marine sponge Callyspongia sp. from the Red Sea. The structure elucidation of the isolated compounds were determined based on detailed spectroscopic data including ¹D and ²D nuclear magnetic resonance (NMR) experimental analyses in combination with high resolution electrospray ionization mass spectrometry (HR-ESI-MS), while the absolute stereochemistry of all amino acids components of nocardiotide A (1) was deduced using Marfey's method. Additionally, ten known metabolites were dereplicated using HR-ESI-MS analysis. Nocardiotide A (1) displayed significant cytotoxic effects towards the murine CT26 colon carcinoma, human HeLa cervix carcinoma, and human MM.1S multiple myeloma cell lines. The results obtained revealed sponge-associated Nocardiopsis as a substantial source of lead natural products with pronounced pharmacological activities.

Volatiles from the fungal microbiome of the marine sponge Callyspongia cf. flammea.

The volatiles emitted by five fungal strains previously isolated from the marine sponge Callyspongia cf. flammea were captured with a closed-loop stripping apparatus (CLSA) and analyzed by GC-MS. Besides several widespread compounds, a series of metabolites with interesting bioactivities were found, including the quorum sensing inhibitor protoanemonin, the fungal phytotoxin 3,4-dimethylpentan-4-olide, and the insect attractant 1,2,4-trimethoxybenzene. In addition, the aromatic polyketides isotorquatone and chartabomone that are both known from Eucalyptus and a new O-desmethyl derivative were identified. The biosynthesis of isotorquatone was studied by feeding experiments with isotopically labeled precursors and its absolute configuration was determined by enantioselective synthesis of a reference compound. Bioactivity testings showed algicidal activity for some of the identified compounds, suggesting a potential ecological function in sponge defence.

Diketopiperazines from Costa Rican endolichenic fungus Colpoma sp. CR1465A.

Three new diketopiperazines (1-3), cyclo(l-Pro-d-trans-Hyp) (1), cyclo(l-Pro-d-Glu) (2), and cyclo(d-Pro-d-Glu) (3) and five known diketopiperazines (4-8) were isolated from the endolichenic fungus Colpoma sp. CR1465A identified from the Costa Rican plant Henriettea tuberculosa (Melatomataceae). The structures of the new compounds 1-3 were elucidated using a combination of extensive spectroscopic analyses, including 2D NMR and HR-MS, and their absolute configurations were determined by a combination of NOESY analysis and Marfey's method. Cyclo(l-Pro-d-allo-Thr) (4) was recently isolated from a South China Sea marine sponge Callyspongia sp., but its NMR spectroscopic data were not reported, and cyclo(l-Pro-l-Asp) (5) was previously reported but only as a synthetic product. The NMR data assignments of compounds 4 and 5 are reported for the first time. All of the isolated compounds were tested for antifungal and antimicrobial properties.

Callyazepin and (3R)-Methylazacyclodecane, Nitrogenous Macrocycles from a Callyspongia sp. Sponge.

Callyazepin (1) and (3R)-methylazacyclodecane (2), nitrogenous macrocycles, were isolated from a tropical Callyspongia sp. sponge. The combined spectroscopic analyses revealed that the structure of 1 is a bicyclic azepane ammonium salt of a novel structural class derived from mixed biogenetic origins. The configuration of the whole molecule and the conformation of the formamide group were assigned by proton-proton coupling constants, a NOESY analysis, and the application of the phenylglycine methyl ester method. The structure of 2 was identified using combined spectroscopic analyses and ECD measurements. These compounds exhibited moderate cytotoxic activities against the K562 and A549 cell lines.

Callyaerins from the Marine Sponge Callyspongia aerizusa: Cyclic Peptides with Antitubercular Activity.

Chemical investigation of the Indonesian sponge Callyspongia aerizusa afforded five new cyclic peptides, callyaerins I-M (1-5), along with the known callyaerins A-G (6-12). The structures of the new compounds were unambiguously elucidated on the basis of one- and two-dimensional NMR spectroscopy and mass spectrometry. In addition, the structures of callyaerins D (9), F (11), and G (12), previously available in only small amounts, have been reinvestigated and revised. All compounds were tested in vitro against Mycobacterium tuberculosis, as well as against THP-1 (human acute monocytic leukemia) and MRC-5 (human fetal lung fibroblast) cell lines, in order to assess their general cytotoxicity. Callyaerins A (6) and B (7) showed potent anti-TB activity with MIC90 values of 2 and 5 µM, respectively. Callyaerin C (8) was found to be less active, with an MIC90 value of 40 µM. Callyaerin A (6), which showed the strongest anti-TB activity, was not cytotoxic to THP-1 or MRC-5 cells (IC50 > 10 µM), which highlights the potential of these compounds as promising anti-TB agents.

Antichlamydial sterol from the Red Sea sponge Callyspongia aff. implexa.

Marine sponges are rich sources of natural products exhibiting diverse biological activities. Bioactivity-guided fractionation of the Red Sea sponge Callyspongia aff. implexa led to the isolation of two new compounds, 26,27-bisnorcholest-5,16-dien-23-yn-3ß,7α-diol, gelliusterol E (1) and C27-polyacetylene, callimplexen A (2), in addition to the known compound ß-sitosterol (3). The structures of the isolated compounds were determined by 1D- and 2D-NMR techniques as well as high-resolution tandem mass spectrometry and by comparison to the literature. The three compounds (1-3) were tested against Chlamydia trachomatis, an obligate intracellular gram-negative bacterium, which is the leading cause of ocular and genital infections worldwide. Only gelliusterol E (1) inhibited the formation and growth of chlamydial inclusions in a dose-dependent manner with an IC50 value of 2.3 µM.

Epipolythiodiketopiperazines from the Marine Derived Fungus Dichotomomyces cejpii with NF-κB Inhibitory Potential.

The Ascomycota Dichotomomyces cejpii was isolated from the marine sponge Callyspongia cf. C. flammea. A new gliotoxin derivative, 6-acetylmonodethiogliotoxin (1) was obtained from fungal extracts. Compounds 2 and 3, methylthio-gliotoxin derivatives were formerly only known as semi-synthetic compounds and are here described as natural products. Additionally the polyketide heveadride (4) was isolated. Compounds 1, 2 and 4 dose-dependently down-regulated TNFα-induced NF-κB activity in human chronic myeloid leukemia cells with IC50s of 38.5 ± 1.2 µM, 65.7 ± 2.0 µM and 82.7 ± 11.3 µM, respectively. The molecular mechanism was studied with the most potent compound 1 and results indicate downstream inhibitory effects targeting binding of NF-κB to DNA. Compound 1 thus demonstrates potential of epimonothiodiketopiperazine-derived compounds for the development of NF-κB inhibitors.

Esters of the marine-derived triterpene sipholenol A reverse P-GP-mediated drug resistance.

Our previous studies showed that several sipholane triterpenes, sipholenol A, sipholenone E, sipholenol L and siphonellinol D, have potent reversal effect for multidrug resistance (MDR) in cancer cells that overexpressed P-glycoprotein (P-gp/ABCB1). Through comparison of cytotoxicity towards sensitive and multi-drug resistant cell lines, we identified that the semisynthetic esters sipholenol A-4-O-acetate and sipholenol A-4-O-isonicotinate potently reversed P-gp-mediated MDR but had no effect on MRP1/ABCC1 and BCRP/ABCG2-mediated MDR. The results from [3H]-paclitaxel accumulation and efflux studies suggested that these two triterpenoids were able to increase the intracellular accumulation of paclitaxel by inhibiting its active efflux. In addition, western blot analysis revealed that these two compounds did not alter the expression levels of P-gp when treated up to 72 h. These sipholenol derivatives also stimulated the ATPase activity of P-gp membranes, which suggested that they might be substrates of P-gp. Moreover, in silico molecular docking studies revealed the virtual binding modes of these two compounds into human homology model of P-gp. In conclusion, sipholenol A-4-O-acetate and sipholenol A-4-O-isonicotinate efficiently inhibit the P-gp and may represent potential reversal agents for the treatment of multidrug resistant cancers.

Evaluating summary statistics used to test for incomplete lineage sorting: mito-nuclear discordance in the reef sponge Callyspongia vaginalis.

Conflicting patterns of population differentiation between the mitochondrial and nuclear genomes (mito-nuclear discordance) have become increasingly evident as multilocus data sets have become easier to generate. Incomplete lineage sorting (ILS) of nucDNA is often implicated as the cause of such discordance, stemming from the large effective population size of nucDNA relative to mtDNA. However, selection, sex-biased dispersal and historical demography can also lead to mito-nuclear discordance. Here, we compare patterns of genetic diversity and subdivision for six nuclear protein-coding gene regions to those for mtDNA in a common Caribbean coral reef sponge, Callyspongia vaginalis, along the Florida reef tract. We also evaluated a suite of summary statistics to determine which are effective metrics for comparing empirical and simulated data when testing drivers of mito-nuclear discordance in a statistical framework. While earlier work revealed three divergent and geographically subdivided mtDNACOI haplotypes separated by 2.4% sequence divergence, nuclear alleles were admixed with respect to mitochondrial clade and geography. Bayesian analysis showed that substitution rates for the nuclear loci were up to 7 times faster than for mitochondrial COI. Coalescent simulations and neutrality tests suggested that mito-nuclear discordance in C. vaginalis is not the result of ILS in the nucDNA or selection on the mtDNA but is more likely caused by changes in population size. Sperm-mediated gene flow may also influence patterns of population subdivision in the nucDNA.