DNA sequencing to support species-level identification of freshwater sponges in western Pennsylvania

Freshwater sponges perform numerous essential environmental roles, including the ability to serve as sensitive bioindicators of water quality. While 31 freshwater sponge species have been detected across North America, documentation of freshwater species within western Pennsylvania is limited. From our collections conducted in local waters from 2015-2019, we report the presence of three freshwater sponge species, identified as Ephydatia muelleri, Ephydatia fluviatilis, and Heteromeyenia tubisperma. These species were identified and differentiated based on the morphology of spicules- microscopic crystalline skeletal structures that support the sponge’s scaffold and protects them from predation. While these spicules show subtle differences in morphology between species, this method of identification can be subjective and confounded by variation in spicule traits. This is prevalent especially in closely related sister species.

In order to validate morphological identifications, we isolated genomic DNA from our freshwater sponge samples and used PCR to amplify and sequence a 660bp hyper-variable region of the mitochondrial cytochrome oxidase I (COI) gene. Alignment of our sample sequences to reference sequences from GenBank of E. muelleri and E. fluviatilis revealed two variable sites between them, which may be sufficient to discriminate between the congeners, even though the reference sequences presented some intraspecies inconsistencies. Comparison of our sample sequences suggested five sites at which H. tubsiperma was variable from the two Ephydatia species, but no GenBank reference sequences were available for H. tubisperma. Overall, the short variable sequence amplified with these mitochondrial COI primers appears to be a promising sequence region to support morphological identifications and might also be a useful region for detecting and identifying freshwater sponges from environmental DNA water samples.

Figure 2: The region of the COI gene amplified in this study is compared in parallel for Ephydatia muelleri, Ephydatia fluviatilis, and Heteromeyenia tubisperma in order to observe intra-species differentiation at possible diagnostic sites. The forward and reverse primers, produced by Lavrov et al15, are shown at their respective alignments on COI. The base pair sites at which potential species diagnostic variation is observed between the three species within the experimental sequences (Table 2) are included on the gene as base pair numbers. The associated base pair for the variable site is shown for each species. Coordinating boxes were used to highlight the specific unique base pair that suggests the site to be diagnostic for the species sample. Sample size for number of individuals collected-extracted-amplified-sequenced are included in parentheses following the corresponding species name.

Results

Table 2: A comparison of available GenBank reference sequences and the base pairs present at potential species diagnostic sites among seven sequenced samples of the samples collected in western Pennsylvania. Morphological identification is included with the sequenced samples. For E. muelleri and E. fluviatilis, BLAST confirmed the morphological identification. One species diagnostic site was identified between E. muelleri and E. fluviatilis, indicated by a single asterisk: 504. Four additional variable sites were included among these two species due to discrepancy between the reference sequences: sites 477 and 640 (JN209966.1 and DQ176777.1 disagree); sites 717 and 726 (EU237481.1 and DQ176778.1 disagree). Variability in the forward and reverse chromatograms at site 745 suggested a polymorphic haplotype for sample SB. Five possible diagnostic sites exist between H. tubispermaand the Ephydatia species, indicated by a double asterisk: 534, 547, 568, 612, 687.

Emily Simon1, Marc Yergin2, Brady Porter1; 1Department of Biological Sciences, Duquesne University, Pittsburgh, PA; 2Carnegie Museum of Natural History, Pittsburgh, PA

References

Abstract

1. National Park Service. (2017). Freshwater Sponges. Retrieved 2019.2. Thorp, J. H., & Rogers, C. D. (2016). Keys to Neartic Fauna (Vol. 2). San Diego: Academic Press.3. Manconi, R., & Pronzato, R. (2008). Global diversiy of sponges (Porifera: Spongillina) in freshwater. Hydrobiologia(595), 27-33.4. Rader, R. (1984). Factors Affecting Distribution of a Freshwater Sponge. Freshwater Invertebrate Biology, 3(2), 86.5. Kahlert, M., & D., N. (1997). Archiv fur Hydrobiologie, 69-81.6. Benfey, T. J., & Reiswig, H. M. (1982). Temperature, pH, and photoperiod effects upon gemmule hatching in the freshwater sponge, Ephydatia mülleri (Porifera, Spongillidae). Journal of Experimental Zoology, 221(1).7. Paulsen, T.S., Hoback, W.W. (2011). A Report of Freshwater Sponge (Porifera: Spongillidae) in Central Nebraska. Transactions of Nebraska Academy of Sciences and Affiliated Societies, 6.8. Reindhart, C.H. (1999). Acid Mine Drainage in Pennsylvania Strems: Ironing Out the Problem. Restoration and Reclamation Review, 5(1)9. Potts, E. (1887). Contributions Towards a Synopsis of the American Forms of Fresh Water Sponges with Descriptions of Those Named by Other Authors and from All Parts of the World. Proceedings of the Academy of Philadelphia, 39(2), 158-279.10. Wurtz, C. B. (1950). Sponges of Pennsylvania and Adjacent States. Notulae Naturae of the Academy of Natural Sciences of Philadelphia.11. Raslins, J. E., & Bier, C. W. (1998). Invertebrates: Review of status

in Pennsylvania. Inventory and Monitoring of Biotic Resources in Pennsylvania, 1, 85-120.National Museum of Natural History. (2019). Department of Invertebrate Zoology Collections.12. Manconi, R., & Pronzato, R. (2016). Phylum Porifera. In J. H. Thorp, & C. D. Rogers, Keys to Neartic Fauna. San Diego.13. Andus, S., Tubic, B., Ilic, M., Duknic, J., Gacic, Z., & Paunovic, M. (2016). Freshwater Sponges – Skeletal Structure Analysis Using Light Microscopy and Scanning Electron Microscopy. Water Research and Management, 6(2), 15-17. 14. Yergin, M.L., Pearce, T.L., Simon, E.F. (Submitted 2019). First Report of Three Freshwater Sponges (Porifera: Demospongia: Spongillidae) in Western Pennsylvania (Ohio River Watershed). Proceedings of the Academy of Natural Sciences of Philadelphia-Unpublished.15. Lavrov, D.V., wang, X., & Kelly, M. (2008). Reconstructing ordinal relationships in Demospongiae using mitochondrial genomic data. Molecular Phylogenetics and Evolution, 49(1), 111-124.16. Pennsylvania Department of Environmental Protection. (2019). Agriculture. Retrieved 2019.17. Nine Mile Run Watershed Association. 2016. Retrieved 2019.18. Frost, T.M. 2001. Ecology and Classification of North American Freshwater Invertebrates, 2nd edition. Thorp, J.H., Covich, A.P, editors. Academic Press

• Approximately 30 species of freshwater sponges (family Spongillidae) can be found in flowing waters around North America• Provide environmental services: nutrient cycling; water filtration; source of food to aquatic

invertebrates; microbial microhabitats• Valuable bioindicators: inhibited by high levels of nitrate and other contaminants

• Very little information exists on freshwater sponge conservation and population status in western Pennsylvania• Four previous studies documenting sponge presence in the state of Pennsylvania (primarily

eastern)• Total of 16 individual species identified between 1887-2016

• Current method of species-level identification: Morphological• Skeletal structures made of siliceous spicules- megascleres, microscleres, gemmuloscleres• ID using geometry, size, and shape of the different spicule types present• Issues distinguishing between closely related species; subjective interpretation; lack of

morphological references• This study examines the utility of DNA sequences from the mitochondrial cytochrome

oxidase I gene (COI) of freshwater sponges as a complementary procedure to morphological identification• Observe specific COI base-pair variation between three species that were collected by us in

western Pennsylvania sites between 2015-2019 (Ephydatia muelleri, Ephydatia fluviatilis, andHeteromeyenia tubisperma)

We hypothesize that sufficient species-specific variation exists in this region of the COI gene for species-level identification of our freshwater sponge samples, and that this variation will consistently correspond to

the morphological identifications.

Introduction

AcknowledgementsWe thank the Carnegie Museum of Natural History and the Department of Biological Sciences at Duquesne University for their support. We thank the Bayer School of Natural and Environmental Sciences for financial support. We appreciate the guidance of Dr. Timothy Pearce, Curator of Mollusks at the Carnegie Museum. Laura Howell and Dr. John Stolz assisted with SEM imaging at Duquesne University. We also thank members of the Porter lab including Ashton Callipare, Brandon Hoenig, and Dr. Beth Dakin, for their support and edits.

Figure 1: Morphology of three collected sponge samples; Ephydatiafluviatilis, Ephydatia muelleri, and Heteromeyenia tubisperma. Length and width of megascleres and gemmuloscleres, and microscleres if present, were noted under light microscopy and scanning electron microscopy. Dimensions and geometry were compared to reference measurements from Manconi and Pronzato (2016) in order to identify the species in Yergin et al.14

Column 1 (left) shows E. fluviatilis, collected from Conneaut Lake in Crawford County (11/2017), column 2 (middle) shows E. muelleri, collected from Kooser Run in Somerset county (9/2016), and column 3 (right) shows Heteromeyenia tubisperma, collected from Woodcock Creek in. In vivo images, megascleres (and microscleres as well for H. tubisperma), and gemmuloscleres are shown for each species. Morphological features of interest are highlighted for the in vivo pictures. In the E. fluviatilis sample, spherical, golden colored gemmules are visible. For the E. meulleri sample, large osculum and symbiotic algae are visible. For H. tubisperma, gemmuloscleres are also obvious. This morphological data provided an original species-level identification to which genetic data could be compared.

Species ID

AMorphological ID

Yergin et al 14

BCollectionMolecular ID

C

6%NaOCl

Compare measurements and geometry to Manconi

& Pronzato (2016) references

BLAST & align to reference sequences

for ID and comparison

DNA isolation Skeletal isolation

Ampl

ify COI

Conf

irm

Microscopy

Sequencing

Methodology

Table 1: Location data for sites visited during sampling surveys of freshwater sponges in western Pennsylvania. The survey date, site name, county, qualitative silt observations, sponge presence, and species-level identification (provided by the morphological work by Yergin et al20) are included. Silt is considered to be the prevalent environmental factor effecting sponge presence in this region.20

Laurel Caverns is marked with an asterisk due to this site being mentioned in a small entry in the National Museum of Natural History Invertebrate ZooloyDatabase, stating a sponge (Heteromeyeniatentsperma) was found in the early 1900s. The exact coordinates recorded were revisited for our survey.

• Freshwater sponges were collected from multiple localities within western Pennsylvania between 2015-2019, which suggests high water quality at these sites due to their roles as bioindicators• Crusty’s Lake (Lawrence County)• Conneaut Lake and Woodcock Creek (Crawford County)• Kooser Run and Strawberry Lake Quarry (Somerset County)

• Three species identified: Ephydatia muelleri, Ephydatia fluviatilis, and Heteromeyenia tubisperma (not previously identified in PA)• Molecular identification via amplification of the COI gene• E. muelleri and E. fluviatilis confirmed with BLAST and alignments to reference sequences from GenBank• One potential species diagnostic site between the two

• H. tubisperma sample was successfully sequenced and aligned to the other experimental sequences, but no reference sequence information is available • Five potential species diagnostic sites at which both the Ephydatia base pairs were consistent, with only H. tubisperma

varying• A cohesive understanding of western Pennsylvania freshwater health is imperative due to acid mine drainage, farm runoff,

and other pollutants16,17,18

• Can link sponge presence and diversity with water quality as a key contribution to comprehensive freshwater conservation• Future directions:• Address intra-species variation occurring among GenBank reference sequences for Ephydatia muelleri and Ephydatia fluviatilis• Submit our COI sequence of Heteromeyenia tubisperma to GenBank as a reference sequence• If potential species diagnostic sites continue to hold up for additional samples, we can recommend this section of the COI gene for

molecular ID of these species (possibility eDNA barcoding applications in the future)

Discussion