Species Diversity and Mitochondrial DNA Analysis of Sponges (Porifera, Demospongiae) in an Anchialine Cave on the Yucatan Peninsula

The anchialine ecosystem in the southeastern Gulf of Mexico has an unexplored fauna, among the most understudied groups is Porifera, molecular approaches to investigate their biology and evolution remain largely short. To broaden these studies, sponge individuals collected in the anchialine cave “Xcalac” in the Mexican state of Quintana- Roo were analyzed using 18S rRNA sequences from sponge metagenomes. The three individuals studied belong to the Cinachyrella, Xestospongia, and Suberites genera. The mitochondrial (mt) genomes of Cinachyrella sp. n. and Xestospongia sp. n. were 18,493 bp to 19,604 bp in length, containing 14 protein-coding genes, 2 rRNA genes (rrnS and rrnL), and 23–25 tRNA genes, respectively. The phylogenomic analysis showed that Cinachyrella sp. n. had the same gene arrangement as the members of its subclade, including sponge species of the Cinachyrella and Geodia genera. The mt genome of Xestospongia sp. n. contained the same gene arrangement as found in other sponges of the same genus and differed from other genera such as Petrosia and Haliclona by an tRNA (tyrosine, Y1) . Variation in mitochondrial genomes (size, gene content, and gene order) was observed when comparing sampled sponge species from the class Demospongiae to the class Homoscleromorpha. This is the first record of Cinachyrella sp. n and Suberites sp. n. in an anchialine cave on the southeastern Yucatan peninsula, and the first report of the mitochondrial genome analysis of Cinachyrella sp. n. and Xestospongia sp. n., contributing to a better understanding of the diversity and phylogeny of sponges in this ecosystem.

anchialine ecosystem, mitochondrial DNA, 18S rRNA, phylogenomic analysis, Demospongiae 3 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted November 3, 2024. ; https://doi.org/10.1101/2024.10.31.621400doi: bioRxiv preprint Suárez-Moo 3

The Yucatan peninsula is a limestone platform with a subterranean aquifer ecosystem that has unique geological and chemical conditions (Bauer-Gottwein et al. 2011). These conditions have led to the formation of an underwater anchialine cave system approximately 330 km in length (Calderón-Gutiérrez et al. 2017) . This ecosystem can exhibit extensive horizontal branching, connections to the sea through conduits, and increasing depth away from the coastline (Benítez et al. 2019). These caves are characterized by light limitation, pressure and temperature variations, varying levels of dissolved organic matter, and fluctuating oxygen availability (Yañez-Mendoza et al. 2007, Romero 2019). Such physicochemical conditions may result in high species richness and endemism rates reported for these ecosystems (Yañez-Mendoza et al. 2007, Calderón-Gutiérrez et al. 2017, Gómez and Calderón-Gutiérrez 2020) . Previous investigations of the fauna in the anchialine caves of the region have focused on phytoplankton and tychoplankton communities (Sánchez et al. 2002) , Crustacea (Alvarez et al. 2015, Benítez et al. 2019, Álvarez et al. 2023) , Echinodermata (Mejía- Ortíz et al. 2007, Bribiesca-Contreras et al. 2019) , and other groups such as Annelida and Mollusca (Álvarez et al. 2023). A particular group of interest is the phylum Porifera (sponges), which can enhance primary production, participate in biogeochemical cycles, and provide microhabitats for a wide range of organisms (Koukouras et al. 1996; Pérez- Botello and Simões 2021). A high number of sponge species have been reported in the anchialine caves of Cozumel island in the Yucatan peninsula (Calderón-Gutiérrez et al. 2017, 2018, Gómez and Calderón-Gutiérrez 2020) , in coral reefs in the Caribbean Sea and the Gulf of Mexico (Ugalde et al. 2021, Pérez-Botello et al. 2023), and in mangrove roots from the southern Gulf of Mexico (Castellanos-Pérez et al. 2020). Reports of sponges in the anchialine caves of the mainland Yucatan peninsula are scarce, and there is only one study that reported the microbial communities and biotechnological potential of bacteria isolated from a sponge belonging to the genus Xestospongia, collected in the anchialine cave of Xcalak, Quintana Roo, México (Suárez-Moo et al. 2024). In contrast, marine sponges in the tropics have been broadly studied. including topics such as sponge ecology (Wulff 2006, Araya-Vargas et al. 2020) , new species ’ description(Shen et al. 2022, Shilov et al. 2023, Díaz et al. 2024) , diversity of the microbial communities associated to their tissues (Busch et al. 2022) , metabolic 4 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted November 3, 2024. ; https://doi.org/10.1101/2024.10.31.621400doi: bioRxiv preprint Suárez-Moo 4 potential of their microbial communities (Lesser et al. 2022) , and biotechnological applications of their isolated bacteria (Jayatilake et al. 1996, Su et al. 2014, Santos et al. 2015). A common molecular marker used to determine the phylogenetic relationships among different animals is the mitochondrial DNA (mtDNA). MtDNA-based phylogenetic trees are considered valuable tools for understanding evolutionary history within and between most groups of metazoans (Feng et al. 2023) . Mitochondrial genome analysis has been used to understanding the phylogeny (Zhang et al. 2016, Taboada et al. 2018) , mtDNA evolution (Wang and Lavrov 2008) and divergence time (Plese et al. 2021) of sponges in different environments. The lack of information about the biodiversity and genomic characterization of the fauna from anchialine caves in the Yucatan peninsula is concerning due to the ir important ecological roles in the karst ecosystem and to the po tential changes in th eir diversity in the current c ontext of climate change . Additionally, the region is experiencing increas ing human activity impacts due to the expansion of tourism and urban development. In this study, we describe three sponge species ( Cinachyrella sp. n., Xestospongia sp. n., and Suberites sp. n.) from an anchialine cave on the coastal area in the Yucatan peninsula, including new records for two of the se species. We also provide the annotated mitochondrial genomes of Cinachyrella sp. n. and Xestospongia sp. n., compare their gene arrangements with those of closely related species, and discuss the phylogenetic framework for these sponges using the nucleotide of their respective amino acid sequences

Sample collection and preservation One specimen from each sponge species belonging to the class Demospongiae was collected at a depth of approximately 12 meters inside an anchialine cave (“Cayo Judio”, Xcalak) from the underground karst aquifer sinkhole in the Yucatan Peninsula in September 2020, ( Figure 1; for details see Suarez-Moo et al. (Suárez-Moo et al. 2024)). The sponges were photographed, removed from the nodule with a scalpel, preserved in RNALater, and transported to the lab at 4 °C and immediately stored at 5 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted November 3, 2024. ; https://doi.org/10.1101/2024.10.31.621400doi: bioRxiv preprint Suárez-Moo 5 −20 °C until DNA extraction. The sponges’ samples were identified using a traditional taxonomic approach based on the tissues and spicules (Gómez and Calderón-Gutiérrez 2020). The samples w ere collected under the Secretaría de Agricultura, Ganadería, Desarrollo Rural, Pesca y Alimentación (SAGARPA) official permit number PPF/DGOPA-062/21. DNA extraction and illumina sequencing Genomic DNA from approximately 1 cm³ of the sponge tissues was extracted in triplicate using a ZymoBIOMICS Miniprep D4300 Kit (Zymo Research, Irvine, USA). DNA from the triplicates was pooled, and its purity and concentration were analyzed using a spectrophotometer (Nanodrop 2000, Thermo Fisher Scientific, Waltham, USA) and visualized by agarose gel electrophoresis. The DNA was stored at –20 °C for further processing. The extracted DNA was subjected to quality control (Qubit Fluorometer), library preparation, and shotgun sequencing by Novogene (Davis, California, USA), on a NovaSeq. 6000 platform (Illumina). Taxonomic assignation The quality of the raw reads was checked with FastQC version 0.11.2 (Andrews, 2010) and multiQC (Ewels et al. 2016) . Low‐quality bases (per base sequence quality <33) were removed with Trimmomatic version 0.39 (Bolger et al. 2014) . High-quality reads (encompassing prokaryotic and eukaryotic reads) were aligned against a nonredundant version of the SILVA database v138 (Quast et al., 2013) with an E-value <10−5. Sequences matching this database were considered potential rRNA gene fragments, and they were aligned against Eukarya hidden Markov models (HMMs) using SSU-ALIGN v. 0.1.1 (Nawrocki, 2009) to identify true sequences. The Eukarya rRNA reads were assembled in rRNA contigs with metaSPAdes version v. 3.13.0 (Nurk et al. 2017). A mapping of the Eukarya rRNA reads against the different Eukaryotic rRNA contigs from the three sponges samples was performed using the software Bowtie 2 (Langmead and Salzberg 2012). To get an estimate of the mean coverage of each Eukaryotic rRNA contig, the following formula was used: (Depth of coverage) × (Number of bases on contig)/ (Length of the contig). The frequency of the mean coverage of the Eukaryotic 6 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted November 3, 2024. ; https://doi.org/10.1101/2024.10.31.621400doi: bioRxiv preprint Suárez-Moo 6 rRNA contigs detected in each sponge library was used in an alluvial diagram with the package ggplot2 (Wickham 2016). The Eukaryotic rRNA contigs assembled were classified using a BLASTN (Altschul et al., 1997) against the SILVA taxonomy database (Quast et al., 2013) and NCBI database. The contigs that matched the 18S rRNA sequences of the Porifera phylum were extracted, and reference sequences downloaded from the GenBank database (Table S1) were aligned using Mafft v.7.305b (Katoh and Standley 2013) and adjusted with trimAl v1.4.rev15 (Capella-Gutiérrez et al. 2009) . Phylogenetic estimation was performed using IQ-TREE (Nguyen et al. 2015) with 10,000 ultrafast bootstraps and the -m MFP option to find the best model that fitted our data. The phylogenetic tree was visualized with iTOL (Letunic and Bork 2019) . A parallel study of the sponge microbiome was conducted in our lab, therefore the prokaryotic reads and contigs of these metagenomes were not analyzed in this study. The nucleotide sequences of the 18S rRNA contigs assembled in this study were deposited in the NCBI under the accession number PQ492272-PQ492274 Mitochondrial genome assembly and annotation The high-quality reads (encompassing prokaryotic and eukaryotic reads) were used to map reads back to the reference mitochondrial genomes belonging to genera identified in the 18S rRNA analysis (Table S2) using Bowtie 2 (Langmead and Salzberg 2012). SAMtools v.1.9 (Li and Durbin 2009) was used to convert files to binary format for further downstream analyses. These mapped reads were used for the mitogenomes assembly using metaSPAdes version v. 3.13.0 (Nurk et al. 2017) . The mitogenomes were annotated using web server MITOS2 (Bernt et al. 2013) with the Ascidian NCBI code for translation, and subsequently manually curated and visualized with Geneious Prime 2024.0.5 (Biomatters Ltd, Auckland, New Zealand). Transfer RNA genes were identified using the tRNAscan-SE program (Lowe and Eddy 1996) and MITOS2 (Bernt et al. 2013). For the phylogenomic analysis, the nucleotide sequences of all protein-coding genes, along with rrnS and rrnL sequences from the BE1 and BE2 sponge mitogenomes and reference mitogenomes downloaded from the GenBank database (Table S2) were concatenated using custom bioinformatic commands and aligned using Mafft v.7.305b 7 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted November 3, 2024. ; https://doi.org/10.1101/2024.10.31.621400doi: bioRxiv preprint Suárez-Moo 7 (Katoh and Standley 2013) . The length of the alignment was adjusted with trimAl v1.4.rev15 (Capella-Gutiérrez et al. 2009). The phylogenomic tree was performed using IQ-TREE (Nguyen et al. 2015) with 10,000 ultrafast bootstraps and the -m MFP option to find the best model that fitted to our data. The phylogenomic tree was visualized with iTOL (Letunic and Bork 2019).

Taxonomic assignation of the three sponges species from the “Xcalak” anchialine cave The morphology analysis indicated that the s amples BE1, BE2, and BE3 were sponges from the orders Tetractinellida , Haplosclerida, and Suberitida respectively. Using standard identification keys based on spicule analysis, Cinachyrella was identified as the candidate genus for sponge BE1 , Haliclona or Xestospongia were potential candidates for BE2, while the family Suberitidae was identified as the candidate for the spongeBE3 in this study. The taxonomic identification of the three sponges was confirmed through the taxonomic assignment of rRNA contigs assembled from the metagenomic shotgun data. In the taxonomic assignment of the rRNA contigs for the BE1 sponge, five contigs were assembled. The longest contig with the most coverage, (1704 bp) belonged to the genus Cinachyrella (order Tetractinellida, phylum Porifera) (Figure 2A and Table S3). However, other organisms, including sponges from the genus Suberites (with a low-abundance, and short contig) and the genera Erinaceusyllis and Syllis (order Phyllodocida, phylum Annelida), were also associated with the BE1 sponge (Figure 2A and Table S3). Thirteen contigs were assembled for the sponge BE2. Three of them were assigned to the genus Xestospongia (order Haplosclerida, phylum Porifera), including one contig with the highest coverage and a length of 2029 bp (Figure 2A and Table S3). A short contig (171 bp) belonging to the genus Suberites (order Suberitida, phylum Porifera) was also assembled from this sample (Figure 2A and Table S3). Another long contig (1830 bp) was assigned to the genus Diadumene (order Actiniaria, phylum Cnidaria) (Figure 2A and Table S3). The three contigs assembled in the sponge BE3 were assigned to the genus Suberites (order Suberitidae, phylum Porifera), with the longest contig measuring 2048 bp (Figure 2A and Table S3). 8 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted November 3, 2024. ; https://doi.org/10.1101/2024.10.31.621400doi: bioRxiv preprint Suárez-Moo 8 Using a BLASTn against the NCBI database, the 18S rRNA sequence of sponge BE1 had a 99.6% identity with Cinachyrella sp. Sp11 (AY734439.1) and 98.8% with Cinachyrella schulzei (KX894470.1) . The BE2 sponge had 98% identity with Xestospongia sp. USNM 1204834 (KC902312.1) and 93% with X. muta (AY621510.1). The BE3 sponge had a 100% identity with Suberites aurantiacus voucher MNRJ15701 (KC902192.1) and 99.65% with S. massa voucher BELUM:Mc4528 (KC902066.1). Phylogeny and mitochondrial genome analysis The 18S rRNA phylogenetic analysis using reference organisms confirmed taxonomic affiliations and showed that the BE1 s ponge clustered with species of the genus Cinachyrella, BE2 clustered with those from Xestospongia, and the BE3 sponge with Suberites (Figure 2B). Evolutionary speaking, sponge BE1 was more closely related to sponge BE3 than to the BE2 sponge. Therfore, based on the taxonomic assignment of the rRNA contigs and 18S rRNA phylogenetic analyses, the sponges BE1, BE2, and BE3 were conservatively identified as Cinachyrella sp. n., Xestospongia sp. n., and Suberites sp. n., respectively. In this study, only the mt genomes of Cinachyrella sp. n. and Xestospongia sp. n. were used, as Suberites sp. n. sequences had a low-quality assembly characterized by a small number of short contigs, and poor gene characterization. The mt genome of Cinachyrella sp. n. consists of 18,493 bp (Figure 3A) and includes 23 tRNA genes, 2 rRNA (rrnS and rrnL), and 14 protein coding genes. These include three subunits of the ATP synthase (atp6, atp8, atp9), seven NADH dehydrogenase subunits (nad1, nad2, nad3, nad4, nad4l, nad5, nad6), one apocytochrome b (cob), and three Cytochrome C oxidase subunits (cox1, cox2, cox3) (Figure 3A). All genes are positioned on the heavy strand and are transcribed counter-clockwise. The mt genome of Xestospongia sp. consists of 19,604 bp (Figure 3B) and contains 25 tRNA genes, 2 rRNA (rrnS and rrnL), and 14 protein coding genes, including three subunits of the ATP synthase (atp6, atp8, atp9), seven NADH dehydrogenase subunits (nad1, nad2, nad3, nad4, nad4l, nad5, nad6), one apocytochrome b (cob), and three Cytochrome C oxidase subunits (cox1, cox2, cox3)(Figure 3B). All genes are positioned on the heavy strand and are transcribed clockwise. 9 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted November 3, 2024. ; https://doi.org/10.1101/2024.10.31.621400doi: bioRxiv preprint Suárez-Moo 9 The phylogenomic tree of the concatenated nucleotide alignment of all protein- coding gene sequences, along with rrnS and rrnL shows two clades (Figure 4A): Clade (A) includes the three sponge species belonging to the class Homoscleromorpha (groups G1 and G2), while clade B contains two subclades. The first subclade includes Plenaster craigi (group G3) and species from the genera Geodia and Cinachyrella, including sponge BE1 Cinachyrrella sp. n. (group G4). The second subclade includes species from the genera Petrosia and Haliclona (group G5) and the genus Xestospongia (group G6) including sponge BE2 Xestospongia sp. n. The mt genome of sponge BE1 Cinachyrella sp. n. exhibits the same genome organization and gene arrangement as other sponges from the same genus but also from the genus Geodia (group G4, Figure 4B). In contrast, sponge BE 2 Xestospongia sp. n. shows the same genome organization and gene arrangement as found in other sponges of the same genus and lacks a tRNA (tyrosine, Y1) found in other genera such as Petrosia and Haliclona (group G5 vs group G6, Figure 4B). Groups G4 and G6, to which Cinachyrella sp. n. and Xestospongia sp. n belong , respectively, differ in gene order and the former lacks the trnM gene found in the latter. Variation in the size, gene content, and gene order was observed between mitochondrial genomes of sponge species from the classes Demospongiae and Homoscleromorpha (Figure 4B). Oscarella carmella (group G1, Homoscleromorpha) has the highest number of tRNAs, with 28, while Corticium candelabrum and Plakortis simples (group G2, Homoscleromorpha) have the lowest number, with 5 tRNAs (Figure 4B). In contrast, species belonging to Demospongiae (groups G3–G6) have 14 protein-coding genes and 2 rRNAs (rrnS and rrnL), with a range of 22–25 tRNAs (Figure 4B).

Our study represents the first record of the Cinachyrella sp. n. and Suberites sp. n. in the anchialine system of the Yucatan peninsula , and following the report of Cinachyrella sp. n. and Xestospongia sp. n. ( Suárez-Moo et al. 2024) , this is the first m itochondrial genome analysis for both species . Most studies on the characterization of sponges in Mexico’s anchialine caves have been conducted in Cozumel Island (Calderón-Gutiérrez et al. 2017, 2018, Gómez and Calderón-Gutiérrez 2020) , highlighting the need to 10 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted November 3, 2024. ; https://doi.org/10.1101/2024.10.31.621400doi: bioRxiv preprint Suárez-Moo 10 continue exploring the species diversity and the distribution of sponges in these unusual cave systems from the mainland of the Yucatan Peninsula. The phylum Porifera is highly diverse in anchialine caves such as “La Quebrada” and “El Aerolito”, located in Cozumel Island, with 15 and 29 sponges species reported, respectively, (Table S4). The Xcalak cave only had three sponge genera at the moment it was sampled: Cinachyrella sp. n., Xestospongia sp. n., and Suberites sp. n. with the last two of these being new to the anchialine system, since Cinachyrella kuekenthali ) was previously reported at “La Quebrada” (Calderón- Gutiérrez et al. 2018; Gómez-Gutiérrez and Calderón-Gutiérrez 2020). In total, 161 sponge species have been reported from Mexican waters in the Gulf of Mexico (i.e. not from a karstik anchialine system) (Ugalde et al. 2021), including species from the genera Cinachyrella ( C. alloclada , C. apion , and C. kuekenthali ), Xestospongia ( X. arenosa and X. muta), and Suberites (S. aurantiacus) (Pérez-Botello and Simões 2021; Ugalde et al. 2021). Differences in the number of sponge species between anchialine caves from Cozumel island and the mainland of the Yucatan Peninsula could be related to variation in cave morphological characteristics. The caves “La Quebrada” and “El Aerolito” are significantly longer, with lengths of 9.2 km and 18 km, respectively (Calderón-Gutiérrez et al. 2017, 2018; Gómez-Gutiérrez and Calderón-Gutiérrez 2020), whereas the length of Xcalak cave has been estimated to be only ~30 m (divers’ personal observations). Another important difference is that both Cozumel island caves have entrances connected to the Caribbean Sea and coastal sinkholes locally called “cenotes” (Calderón-Gutiérrez et al. 2017, 2018; Gómez-Gutiérrez and Calderón- Gutiérrez 2020), while the cave at Xcalak has a single entrance, a coastal sinkhole known as “Cayo Judío”. Therefore, the anchialine caves could have an energy input to the anchialine system by a mangrove in the main entrance (El Aerolito cave), and the direct connection with the sea leading to an exceptional biodiversity (Calderón- Gutiérrez et al. 2017). On the other hand, this connection to the Caribbean Sea may enhance species diversity in 'La Quebrada' and 'El Aerolito' caves by transporting marine larvae, including those from deep-sea species, into the anchialine caves via upwelling events (Harmelin & Vacelet 1997) or passively through sea-level changes (Hart Jr. et al. 1985). The presence of open-reef or deep-sea sponges in 'La Quebrada' supports the idea of such connections (Gómez-Gutiérrez and Calderón-Gutiérrez 2020). 11 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted November 3, 2024. ; https://doi.org/10.1101/2024.10.31.621400doi: bioRxiv preprint Suárez-Moo 11 Increased sampling efforts in more mainland anchialine caves can lead to the discovery of new sponge species and increase our knowledge of their biodiversity and biogeography. With the exception of the sponge B3 Suberites sp. n., sponges from Xcalak showed rRNA contigs from non-sponge organisms. Cinachyrella sp. n. had contigs associated with polychaetes (Annelida) of the genera Erinaceusyllis and Syllis, while in Xestospongia sp. n., a long contig was assigned to the genus Diadumene (Cnidaria). Numerous studies have reported symbiotic relationships between sponges and their associated fauna (Koukouras et al. 1996; Ávila et al. 2007; Wulff 2012; Maldonado et al. 2017; Yu et al. 2020). Polychaetes of the genera Erinaceusyllis and Syllis have been reported in the anchialine cave of Aerolito on Cozumel Island (Calderón-Gutiérrez et al. 2017). Although the genus Diadumene has not been reported in anchialine caves of the Yucatan Peninsula, other organisms from the phylum Cnidaria have been recorded (Calderón-Gutiérrez et al. 2017, 2018). The three-dimensional habitat heterogeneity in sponges, their structural complexity, and their ability to synthesize toxic substances may create environments where other species can live in, or gain an adaptive advantage (Pérez-Botello and Simões 2021). Therefore, sequences of non-sponge organisms found within the sponges suggest a symbiotic relationship, however, this topic requires further exploration. The tree topologies based on nuclear rRNA and mitochondrial genes were similar and showed that the sponge BE1 and the sponge BE2 were closely related to sponge species belonging to the genera Cinachyrella and Xestospongia (Demospongiae class). Sponge BE3 was closely related to a marine sponge species of the genus Suberites (18S rRNA phylogenetic analysis). Phylogenetic analyses showed Cinachyrella sp. n. clustered more closely with Suberites sp. n. than to Xestospongia sp. n. suggesting closer evolutionary histories between the first two sponges This observation has been previously highlighted in a wide analysis of 68 taxa belonging to Class Demospongiae (Plese et al. 2021). The total number and order of genes observed in the mt- genomes of Cinachyrella sp. n. and Xestospongia sp. n. were very similar to those found in other demosponge mt genomes (Wang and Lavrov 2008, Taboada et al. 2018) . However, slight differences were observed in the number and arrangement of tRNAs. Identical 12 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted November 3, 2024. ; https://doi.org/10.1101/2024.10.31.621400doi: bioRxiv preprint Suárez-Moo 12 mitochondrial gene order patterns have been reported at the order level in the class Demospongiae (Plese et al. 2021) , and respiratory genes such as nad2-nad5 and atp6- cox3, detected in Cinachyrella sp. n. and Xestospongia sp. n., have been previously reported as highly conserved (Rosengarten et al. 2008) . In this study, analyzed groups belonging to the class Demospongiae (G3–G6) had the same number of rRNAs and in proximity to each other (separated by 1–2 tRNA genes) with the gene order being rrnS- trnG-trnV-rrnL, as reported for several demosponge species (Wang and Lavrov 2008, Taboada et al. 2018). Future research should focus on describing the biodiversity and phylogenetic relationship of anchialine sponges, using molecular approaches (nuclear and mitochondrial DNA) that lead to better classification and comparison between sponges that can inhabit different environments, such as anchialine caves, seawater (Caribbean sea) and Brackish water. Acknowledgments We would like to thank the Posgrado en ciencias del mar y limnología (PCML-UNAM) and CONAHCYT for their support of this project. We want to thank Efraín Chávez Solís, Erick Sosa Rodríguez, and Luis A. Liévano Beltrán for their invaluable support while navigating the incredible depths and lengths of the anchialine cave system. Our appreciation also goes to Miguel A. Acuapan Acosta for his help with drawings. Funding This work was supported by the following grants: Consejo Nacional de Humanidades, Ciencias y Tecnologías (CONAHCyT) Ciencia Básica (A1-S-10785) to APD, a post- doctoral fellowship to PSM (CVU 362331) and a CONAHCYT doctoral fellowship through the Posgrado en Ciencias del Mar y Limnología, UNAM (ARD, CVU: 711899)

Alvarez F, Iliffe TM, Benitez S, Brankovits D, Villalobos JL (2015) New records of anchialine fauna from the Yucatan Peninsula, Mexico. Check List 11. https://doi.org/10.15560/11.1.1505 13 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted November 3, 2024. ; https://doi.org/10.1101/2024.10.31.621400doi: bioRxiv preprint Suárez-Moo 13 Álvarez F, Durán B, Meacham S (2023) Anchialine Fauna of the Yucatan Peninsula: Diversity and Conservation Challenges. Mexican Fauna in the Anthropocene: 287–301. https://doi.org/10.1007/978-3-031-17277-9_13 Andrews S (2010) FastQC A Quality Control tool for High Throughput Sequence Data. Available from: http://www.bioinformatics.babraham.ac.uk/projects/fastqc/.%0A30. Araya-Vargas A, Busutil L, García-Rojas A, Pereira Chaves JM, Piedra-Castro L (2020) Evaluación de la estructura comunitaria de las esponjas marinas en parches arrecifales del Caribe sur, Costa Rica. Boletín de Investigaciones Marinas y Costeras 49: 39–62. https://doi.org/10.25268/bimc.invemar.2020.49.1.773 Ávila E, Carballo JL, Cruz-barraza JA (2007) Symbiotic relationships between sponges and other organisms from the Sea of Cortes ( Mexican Pacific coast ): same problems , same solutions. Porifera Research: Biodiversity, Innovation and Sustainability: 147– 156. Bauer-Gottwein P, Gondwe BRN, Charvet G, Marín LE, Rebolledo-Vieyra M, Merediz-Alonso G (2011) Review: The Yucatán Peninsula karst aquifer, Mexico. Hydrogeology Journal 19: 507–524. https://doi.org/10.1007/s10040-010-0699-5 Benítez S, Iliffe TM, Quiroz-Martínez B, Alvarez F (2019) How is the anchialine fauna distributed within a cave? A study of the Ox Bel Ha System, Yucatan Peninsula, Mexico. Subterranean Biology 31: 15–28. https://doi.org/10.3897/subtbiol.31.34347 Bernt M, Donath A, Jühling F, Externbrink F, Florentz C, Fritzsch G, Pütz J, Middendorf M, Stadler PF (2013) MITOS: Improved de novo metazoan mitochondrial genome annotation. Molecular Phylogenetics and Evolution 69: 313–319. https://doi.org/10.1016/j.ympev.2012.08.023 14 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted November 3, 2024. ; https://doi.org/10.1101/2024.10.31.621400doi: bioRxiv preprint Suárez-Moo 14 Bolger AM, Lohse M, Usadel B (2014) Trimmomatic: A flexible trimmer for Illumina sequence data. Bioinformatics 30: 2114–2120. https://doi.org/10.1093/bioinformatics/btu170 Bribiesca-Contreras G, Pineda-Enríquez T, Márquez-Borrás F, Solís-Marín FA, Verbruggen H, Hugall AF, O’Hara TD (2019) Dark offshoot: Phylogenomic data sheds light on the evolutionary history of a new species of cave brittle star. Molecular Phylogenetics and Evolution 136: 151–163. https://doi.org/10.1016/j.ympev.2019.04.014 Busch K, Slaby BM, Bach W, Boetius A, Clefsen I, Colaço A, Creemers M, Cristobo J, Federwisch L, Franke A, Gavriilidou A, Hethke A, Kenchington E, Mienis F, Mills S, Riesgo A, Ríos P, Roberts EM, Sipkema D, Pita L, Schupp PJ, Xavier J, Rapp HT, Hentschel U (2022) Biodiversity, environmental drivers, and sustainability of the global deep-sea sponge microbiome. Nature Communications 13. https://doi.org/10.1038/s41467-022-32684-4 Calderón-Gutiérrez F, Sánchez-Ortiz CA, Huato-Soberanis L (2018) Ecological patterns in anchialine caves. PLoS ONE 13: 1–19. https://doi.org/10.1371/journal.pone.0202909 Calderón-Gutiérrez F, Solís-Marín FA, Gómez P, Sánchez C, Hernández-Alcántara P, Álvarez-Noguera F, Yáñez-Mendoza G (2017) Mexican anchialine fauna — With emphasis in the high biodiversity cave El Aerolito. Regional Studies in Marine Science 9: 43–55. https://doi.org/10.1016/j.rsma.2016.11.001 Capella-Gutiérrez S, Silla-Martínez JM, Gabaldón T (2009) trimAl: A tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics 25: 1972–1973. https://doi.org/10.1093/bioinformatics/btp348 Castellanos-Pérez P de J, Vázquez-Maldonado LE, Ávila E, Cruz-Barraza JA, Canales- Delgadillo JC (2020) Diversity of mangrove root-dwelling sponges in a tropical coastal 15 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted November 3, 2024. ; https://doi.org/10.1101/2024.10.31.621400doi: bioRxiv preprint Suárez-Moo 15 ecosystem in the southern Gulf of Mexico region. Helgoland Marine Research 74. https://doi.org/10.1186/s10152-020-00545-6 Díaz JA, Ordines F, Massutí E, Cárdenas P (2024) From caves to seamounts: the hidden diversity of tetractinellid sponges from the Balearic Islands, with the description of eight new species. PeerJ 12. https://doi.org/10.7717/PEERJ.16584 Ewels P, Magnusson M, Lundin S, Käller M (2016) MultiQC: Summarize analysis

for multiple tools and samples in a single report. Bioinformatics 32: 3047–3048. https://doi.org/10.1093/bioinformatics/btw354 Feng H, Lv S, Li R, Shi J, Wang J, Cao P (2023) Mitochondrial genome comparison reveals the evolution of cnidarians. Ecology and Evolution 13: 1–16. https://doi.org/10.1002/ece3.10157 Gómez P, Calderón-Gutiérrez F (2020) Anchialine cave-dwelling sponge fauna (Porifera) from La Quebrada, Mexico, with the description of the first Mexican stygobiont sponges. Zootaxa 4803: 125–151. https://doi.org/10.11646/zootaxa.4803.1.7 Harmelin, J.G. & Vacelet, J. (1997) Clues to deep-sea biodiversity in a nearshore cave. Vie et Milieu, 47, 351–354. Hart Jr., C.W., Manning, R.B. & Iliffe, T.M. (1985) The fauna of Atlantic marine caves: evidence of dispersal by sea floor spreading while maintaining ties to deep waters. Proceedings of the Biological Society of Washington, 98, 288–292. Jayatilake GS, Thornton MP, Leonard AC, Grimwade JE, Baker BJ (1996) Metabolites from an antarctic sponge-associated bacterium, Pseudomonas aeruginosa. Journal of Natural Products 59: 293–296. https://doi.org/10.1021/np960095b 16 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted November 3, 2024. ; https://doi.org/10.1101/2024.10.31.621400doi: bioRxiv preprint Suárez-Moo 16 Katoh K, Standley DM (2013) MAFFT multiple sequence alignment software version 7: Improvements in performance and usability. Molecular Biology and Evolution 30: 772– 780. https://doi.org/10.1093/molbev/mst010 Koukouras A, Russo A, Voultsiadou-Koukoura E, Arvanitidis C, Stefanidou D (1996) Macrofauna associated with sponge species of different morphology. Marine Ecology 17: 569–582. https://doi.org/10.1111/j.1439-0485.1996.tb00418.x Langmead B, Salzberg SL (2012) Fast gapped-read alignment with Bowtie 2. Nature

9: 357–359. https://doi.org/10.1038/nmeth.1923 Lesser MP, Sabrina Pankey M, Slattery M, Macartney KJ, Gochfeld DJ (2022) Microbiome diversity and metabolic capacity determines the trophic ecology of the holobiont in Caribbean sponges. ISME Communications 2: 1–12. https://doi.org/10.1038/s43705-022-00196-3 Letunic I, Bork P (2019) Interactive Tree of Life (iTOL) v4: Recent updates and new developments. Nucleic Acids Research 47: 256–259. https://doi.org/10.1093/nar/gkz239 Li H, Durbin R (2009) Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25: 1754–1760. https://doi.org/10.1093/bioinformatics/btp324 Lowe TM, Eddy SR (1996) TRNAscan-SE: A program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Research 25: 955–964. https://doi.org/10.1093/nar/25.5.0955 Maldonado M, Aguilar R, Bannister RJ, Bell JJ, Conway KW, Dayton PK, Díaz C, Gutt J, Kelly M, Kenchington ELR, Leys SP, Pomponi SA, Rapp HT, Rützler K, Tendal OS, Vacelet J, Young CM (2017) Sponge grounds as key marine habitats: A synthetic review of types, structure, functional roles, and conservation concerns. In: Rossi S, Bramanti L, Gori A, Orejas C (Eds) Marine animal forests the ecology of benthic 17 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted November 3, 2024. ; https://doi.org/10.1101/2024.10.31.621400doi: bioRxiv preprint Suárez-Moo 17 biodiversity hotspots. Springer Nature, Cham, Switzerland, 39 pp. https://doi.org/10.1007/978- 3-319-17001-5_24-1 Mejía-Ortíz LM, Yánez G, López-Mejía M (2007) Echinoderms in an anchialine cave in Mexico. Marine Ecology 28: 31–34. https://doi.org/10.1111/j.1439-0485.2007.00174.x Nawrocki, E.P. (2009) Structural RNA homology search and align- ment using covariance models. PhD thesis, Washington Univer- sity in Saint Louis, School of Medicine. Nguyen LT, Schmidt HA, von Haeseler A, Minh BQ. 2015. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol Biol Evol 32: 268–274. doi:10.1093/molbev/msu300 Nurk S, Meleshko D, Korobeynikov A, Pevzner PA (2017) MetaSPAdes: A new versatile metagenomic assembler. Genome Research 27: 824–834. https://doi.org/10.1101/gr.213959.116 Pérez-Botello AM, Dáttilo W, Simões N (2023) Geographic range size and species morphology determines the organization of sponge host-guest interaction networks across tropical coral reefs. PeerJ 11: 1–23. https://doi.org/10.7717/peerj.16381 Pérez-Botello AM, Simões N (2021) Sponge-dwelling fauna: a review of known species from the Northwest Tropical Atlantic coral reefs. Biodiversity Data Journal 9: 1–18. https://doi.org/10.3897/BDJ.9.e63372 Plese B, Kenny NJ, Rossi ME, Cárdenas P, Schuster A, Taboada S, Koutsouveli V, Riesgo A (2021) Mitochondrial evolution in the Demospongiae (Porifera): Phylogeny, divergence time, and genome biology. Molecular Phylogenetics and Evolution 155. https://doi.org/10.1016/j.ympev.2020.107011 18 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted November 3, 2024. ; https://doi.org/10.1101/2024.10.31.621400doi: bioRxiv preprint Suárez-Moo 18 Quast, C., Pruesse, E., Yilmaz, P., Gerken, J., Schweer, T., Yarza, P. et al. (2013) The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Research, 41(D1), D590–D596. Romero OR (2019) Biology and ecology of anchialine environments: a review. ANARTIA 29: 7–19. Rosengarten RD, Sperling EA, Moreno MA, Leys SP, Dellaporta SL (2008) The mitochondrial genome of the hexactinellid sponge Aphrocallistes vastus: Evidence for programmed translational frameshifting. BMC Genomics 9: 1–10. https://doi.org/10.1186/1471-2164-9-33 Sánchez M, Alcocer J, Escobar E, Lugo A (2002) Phytoplankton of cenotes and anchialine caves along a distance gradient from the northeastern coast of Quintana Roo, Yucatan Peninsula. Hydrobiologia 467: 79–89. https://doi.org/10.1023/A:1014936714964 Santos OCS, Soares AR, Machado FLS, Romanos MTV, Muricy G, Giambiagi- deMarval M, Laport MS (2015) Investigation of biotechnological potential of sponge- associated bacteria collected in Brazilian coast. Letters in Applied Microbiology 60: 140–147. https://doi.org/10.1111/lam.12347 Shen C, Cheng H, Zhang D, Wang C (2022) Two New Species and One New Genus of Glass Sponges (Hexactinellida: Euplectellidae and Euretidae), From a Transect on a Seamount in the Northwestern Pacific Ocean. Frontiers in Marine Science 9: 1–12. https://doi.org/10.3389/fmars.2022.852498 Shilov VA, Kamenev YO, Semenchenko AA, Kiyashko SI, Mordukhovich V V (2023) New sponge species of the family Vulcanellidae (Demospongiae: Tetractinellida) from the Piip submarine volcano and adjacent areas (Bering Sea, NW Pacific). Deep Sea Research Part II: Topical Studies in Oceanography 208: 105229. https://doi.org/https://doi.org/10.1016/j.dsr2.2022.105229 19 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted November 3, 2024. ; https://doi.org/10.1101/2024.10.31.621400doi: bioRxiv preprint Suárez-Moo 19 Su P, Wang DX, Ding SX, Zhao J (2014) Isolation and diversity of natural product biosynthetic genes of cultivable bacteria associated with marine sponge mycale sp. from the coast of Fujian, China. Canadian Journal of Microbiology 60: 217–225. https://doi.org/10.1139/cjm-2013-0785 Suárez-Moo P, García-martínez NC, Márquez-velázquez NA, Figueroa M, Allen E, Prieto-davó A (2024) Exploring the microbial community and biotechnological potential of the sponge Xestospongia sp . from an anchialine cave in the Yucatán Peninsula. Ciencias Marinas. Taboada S, Kenny NJ, Riesgo A, Wiklund H, Paterson GLJ, Dahlgren TG, Glover AG (2018) Mitochondrial genome and polymorphic microsatellite markers from the abyssal sponge Plenaster craigi Lim & Wiklund, 2017: tools for understanding the impact of deep-sea mining. Marine Biodiversity 48: 621–630. https://doi.org/10.1007/s12526-017- 0786-0 Ugalde D, Fernandez JCC, Gómez P, Lôbo-Hajdu G, Simões N (2021) 5031 Zootaxa An update on the diversity of marine sponges in the southern gulf of Mexico coral reefs. 1–112 pp. https://doi.org/10.11646/zootaxa.5031.1.1 Wang X, Lavrov D V. (2008) Seventeen new complete mtDNA sequences reveal extensive mitochondrial genome evolution within the demospongiae. PLoS ONE 3: 1– 11. https://doi.org/10.1371/journal.pone.0002723 Wickham, H. 2016. ggplot2: Elegant graphics for data analysis. New York: Springer- Verlag. doi:10.1016/j.jrp.2016.10.004 Wulff JL (2006) Ecological interactions of marine sponges. Canadian Journal of Zoology 84: 146–166. https://doi.org/10.1139/z06-019 20 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted November 3, 2024. ; https://doi.org/10.1101/2024.10.31.621400doi: bioRxiv preprint Suárez-Moo 20 Wulff JL (2012) 61 Advances in Marine Biology Ecological Interactions and the Distribution, Abundance, and Diversity of Sponges. 1st ed. Elsevier Ltd., 273–344 pp. https://doi.org/10.1016/B978-0-12-387787-1.00003-9 Yañez-Mendoza G, Zarza-González E, Mejía-Ortíz LM (2007) Sistemas anquihalinos. In: Biodiversidad acuática de la Isla de Cozumel. Universidad de Quintana Roo- CONABIO, 49–70. Yu MC, Dreyer N, Kolbasov GA, Høeg JT, Chan BKK (2020) Sponge symbiosis is facilitated by adaptive evolution of larval sensory and attachment structures in barnacles. Proceedings of the Royal Society B: Biological Sciences 287. https://doi.org/10.1098/rspb.2020.0300 Zhang Y, Sun J, Li X, Qiu JW (2016) The mitochondrial genome of the deep-sea glass sponge Lophophysema eversa (Porifera, Hexacinellida, Hyalonematidae). Mitochondrial DNA 27: 1273–1274. https://doi.org/10.3109/19401736.2014.945552 21 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted November 3, 2024. ; https://doi.org/10.1101/2024.10.31.621400doi: bioRxiv preprint Suárez-Moo 21 Figure 1. Collection site location and sponge specimens. (A) Study area: anchialine cave "Xcalak" (lime diamond), located in the southeast Gulf of Mexico. (B) Photograph of the cave entrance. (C) BE1 sponge. (D) BE2 sponge. (E) BE3 sponge. All sponges were collected in the anchialine cave "Xcalak." 22 630 631 632 633 634 635 636 637 638 639 640 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted November 3, 2024. ; https://doi.org/10.1101/2024.10.31.621400doi: bioRxiv preprint Suárez-Moo 22 Figure 2. Taxonomic identification and phylogenetic analysis of three sponges species collected in the anchialine cave “Xcalak” . (A) The rRNA contigs were taxonomically assigned to genus, order, and phylum levels, represented by different colors. (B) 18S rRNA contigs assembled from the complete metagenomic data were used, along with reference sequences, in a phylogenetic analysis. The circles and the width of each node are Bayesian posterior probabilities. 23 641 642 643 644 645 646 647 648 649 650 651 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted November 3, 2024. ; https://doi.org/10.1101/2024.10.31.621400doi: bioRxiv preprint Suárez-Moo 23 Figure. 3. Genetic map of Cinachyrella sp. n. and Xestospongia sp. n. circular mitochondrial genomes. All genes are positioned on the heavy strand and are transcribed (A) counterclockwise for Cinachyrella sp. n. and (B) clockwise for Xestospongia sp. n. 24 652 653 654 655 25 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted November 3, 2024. ; https://doi.org/10.1101/2024.10.31.621400doi: bioRxiv preprint Suárez-Moo 24 Fig. 4 Phylogenomic analysis and mitochondrial gene arrangements between Cinachyrella sp. n. and Xestospongia sp. n., demosponge species, and outgroup heteroscleromorph . A) Maximum likelihood phylogenetic tree using a concatenated nucleotide alignment of all protein coding gene sequences along with those of rrnS and rrnL. Circles at bases of nodes represent bootstrap support nodes. B) Mitochondrial genome gene order, the complex I (Lime), apocytochrome b (fuchsia), complex IV (Navy), ATP synthase (Green), tRNAs (pink), rRNA genes (Maroon) are shown. 26 656 657 658 659 660 661 27 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted November 3, 2024. ; https://doi.org/10.1101/2024.10.31.621400doi: bioRxiv preprint Suárez-Moo 25 Supplementary tables Table S1. Reference 18S rRNA sequences used for the phylogenetic analysis of the Cinachyrella sp. n., Xestospongia sp. n., and Suberites sp. n. Table S2. Reference mitogenomes used for the phylogenomic analysis and mitochondrial gene arrangements between Cinachyrella sp. n., and Xestospongia sp. n., demosponge species, and outgroup heteroscleromorph. Table S3. rRNA contigs assembled from metagenomes of the three sponges species (Cinachyrella sp. n., Xestospongia sp. n., and Suberites sp. n.) with %identity, length, coverage and taxonomic identification. Table S4. Comparison of Sponge Species diversity detected in the Anchialine Caves: La Quebrada, El Aerolito, and Xcalak 28 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 29 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted November 3, 2024. ; https://doi.org/10.1101/2024.10.31.621400doi: bioRxiv preprint