Final Report March 30, 2018
Project Title: Connectivity between shallow and mesophotic ecosystems in Puerto Rico and the US Virgin Islands: using corals and commensal fauna
Principal Invetsigator: Nikolaos Schizas, Department of Marine Sciences, University of Puerto Rico, Mayagüez
Co-Principal Investigator: Monica Alfaro, Department of Biology, University of Puerto Rico Mayagüez
The proposal was approved on January 28, 2014 and by February 18, 2014, research activities commenced. The project R-101-1014 ended June 30, 2017.
Summary of findings: Schizas and Alfaro met all the objectives of the proposal by inferring the population structure of the coral Agaricia lamarcki (Hammerman et al. 2017) and two species from their associated commensal species (Veglia et al. In Review). Three mesophotic workshops were held from 2014 until 2016 and over 30 undergraduate students were involved in sorting small invertebrates from mesophotic samples. This effort has uncovered an extremely diverse of fauna most of it which is unknown. Several new species of crustaceans and mites have been described and dozens more are awaiting formal description. Three graduate students of UPRM were supported from SeaGrant and an MS Thesis was the outcome of this support. In addition to the published and ongoing work we communicated our findings to scientists through tour posters, one conference talk and one invited talk to a Conservation Genomics workshop. We have sent reprints of our work to DNER personnel from the Coral Reef Program, Tania Metz Estrella and Ernesto Diaz. Lastly, thanks to the support from SeaGrant, we were recently funded by NSF to continue our work on the mesophotic reefs, especially after the aftermath of Hurricanes Maria and Irma.
Objective 1 – Infer the population structure of a) the coral Agaricia lamarcki and b) their associated commensal species.
1a - Population structure of the coral Agaricia lamarcki
We met the main objective of the proposal by applying the modified double Restriction Associated DNA sequencing (RAD-seq) method on samples collected from the mesophotic and shallow reefs of Puerto Rico and US Virgin Islands This work formed the Thesis of the MS student Nicholas Hammerman, who defended in December 2016, and a manuscript was published in the journal Coral Reefs:
Hammerman NM, RE Rivera-Vicens, MP Galaska, E Weil, RS Appledoorn, M Alfaro, NV Schizas (2017). Population connectivity of the plating coral, Agaricia lamarcki from southwest Puerto Rico. Coral Reefs DOI: 10.1007/s00338-017-1646-x
A pdf reprint has been submitted to SeaGrant with the Final Report.
Abstract: Identifying genetic connectivity and discrete population boundaries is an important objective for management of declining Caribbean reef-building corals. A double digest restriction associated DNA (ddRAD) sequencing protocol was utilized to generate 321 single nucleotide polymorphisms (SNPs) to estimate patterns of horizontal and vertical gene flow in the brooding Caribbean plate coral, Agaricia lamarcki. Individual colonies (n=59) were sampled from eight locations throughout southwestern Puerto Rico from six shallow (~10-20m) and two mesophotic habitats (~30-40m). Descriptive summary statistics (FST, AMOVA), as well as analysis through Landscape and Ecological Associations (LEA) and Discriminant Analysis of Principal Components (DAPC) estimated high population connectivity with subtle subpopulation structure between all sampling localities within southwestern Puerto Rico.
2b - - Population structure of the associated commensal fauna of the coral Agaricia lamarcki
We met the second part of the main objective of the proposal by using molecular markers to infer connectivity patterns of 2 species exhibiting diverse life histories and associated with the Carbbean mesophotic reefs. This work formed the basis of a manuscript submitted to the Journal of the Marine Biological Association of the United Kingdom and is currently under review:
Veglia AJ, NM Hammerman, CR Rivera, M Lucas, A Galindo Estronza, PH Corgosinho, and NV Schizas Characterizing population structure of coral-associated fauna from mesophotic and shallow habitats in the Caribbean (In review). Journal of the Marine Biological Association of the United Kingdom.
A pdf reprint will be submitted to SeaGrant when it has been accepted by the journal.
Abstract: Symbiotic relationships are a common phenomenon among marine invertebrates, forming both obligatory and facultative dependencies with their host. Here, we investigate and compare the population structure of an obligate symbiont (Ceratoconcha domingensis; a barnacle) on the scleractinian coral Agaricia lamarcki from shallow and mesophotic habitats in southwest Puerto Rico, and a facultative symbiont (Laophontella armata; a harpacticoid copepod) from mesophotic reefs in El Seco, Puerto Rico and mangrove habitats in Curaçao. The barnacles were excavated from shallow (<30 m) and mesophotic (>30 m) colonies of A. lamarcki inhabiting La Parguera and Guánica¸ southwest Puerto Rico. The copepod, L. armata were collected from mesophotic reefs (>50 m) of El Seco, Vieques (east coast of Puerto Rico) and mangrove sediments (<3 m) from Curaçao. The Cytochrome Oxidase Subunit I (COI) gene was amplified and sequenced for barnacle (n=93) and copepod (n=54) specimens. Molecular analyses revealed no population structure between mesophotic and shallow barnacle populations within La Parguera and Guánica (ΦST = 0.0044, p= 0.33). The absence of population structure was expected due to the pelagic naupliar larva of the barnacles and the connectivity patterns exhibited by the coral itself within the same region. In contrast, L. armata was shown to have significant structure between
the mesophotic reef ecosystem of El Seco and mangrove sediments of Curaçao (ΦST = 0.28, p= 0.0). Interestingly, the El Seco and Curaçao copepods had three shared COI haplotypes. Shared haplotypes were unexpected between these sample localities based on the obligatory benthic development of harpacticoid copepods and the geographic distance between the two locations. Three other COI haplotypes from El Seco exhibited higher than expected intra-species variability, potentially representing three new species of harpacticoid copepods or rare, deeply divergent lineages of L. armata. The identification of potentially cryptic species within what is an otherwise easily identified harpacticoid copepod species is evidence of the urgent need for a deeper investigation into the meiofauna diversity associated with mesophotic coral ecosystems (MCEs), as arguably this is the most diverse metazoan component of MCEs
Objective 2 – Highlight the importance and biodiversity of mesophotic reefs through student participation, workshops, community involvement, and publications -Narrative
Biodiversity of Mesophotic reefs
A total of 30 undergraduate students enrolled in research have been working in this project. They analyzed 72 sediment samples collected from the mesophotic reefs of Puerto Rico and US Virgin Islands, separating the organisms from the sediments, sorting and completing the taxonomical classification in major groups. They also participated in all the conferences and regular meetings with PI Schizas and CoPI, Alfaro, where they have received the appropriate training necessary for each step of the sample processing.
The results of their work have provided the following information. A total of 12 benthic taxonomic groups have been identified from collections of the mesophotic reefs. Means and relative abundance of the taxa varied from a minimum of 1,714 to 3,420 ind. The minimum abundances are always found in shallow reefs, followed by intermediate depth reefs. The highest abundances are always found in the mesophotic reefs samples. Copepods are the numerically dominant taxa of the total benthic community with a relative abundance higher than 53%. Amphipods represent the second most abundant assemblage with 15.58% at the deeper stations. The third assemblage is the ostracods with numbers higher than 10% in shallow waters. Tanaids, Isopods and Cumaceans are also more abundant in deeper waters. The results of the Principal Component Analysis (PCA) for the highest numerically taxa from mesophotic coral reefs showed eigenvalues indicating that one component provided 64%, two components explained 88% and three components explained 99.6% of the standardized variance (Figure 3). The first component corresponds to the deep stations, since the first eigenvector shows a very high positive weigh. This component was defined mainly by the abundance pattern of tanaids, isopods and amphipods, which had the highest values for this depth. Also, the ostracods introduced spatial variation, but to a lesser extent, representing the shallow station, the first eigenvector had high positive weight on the shallow stations. Intermediate stations are not well defined since the densities of different taxa are in
between the other two depths. In general terms there is a different community structure for each depth, but certain groups as copepods, the numerically dominant group is found at all depths suggesting vertical migrations and connectivity.
Figure 3. Principal component analysis (PCA) based on the samples abundance of the taxonomic assemblage from Mesophotic Coral Reefs of Puerto Rico and US Virgin Islands.
- SeaGrant PR Supported Table
Category # of new students # of continuing students # of degrees awarded
Sea Grant
Supported MA/MS 2 2 (GalindoEstronza MS, 2015) (Nick Hammerman), MS, 2016
Sea Grant
Supported PhD
Sea Grant
Supported BA/BS
- List of thesis and dissertations by supported students. There are two MS Theses supported by the Seagrant project and both Theses have been completed.
Students Supported (under the academic supervision of PIs Schizas and Alfaro)
1) Alexandra Galindo Estronza, alexandra.galindo@upr.edu, was awarded an MS in Biology under the supervision of PIs Schizas and Alfaro. The theme of Galindo’s Thesis is “Concordance between morphological and genetic barcoding estimates of species diversity in Caribbean benthic ostracods from mesophotic reefs”. Alexandra Galindo defended her Thesis on November 13, 2015 and her manuscript stemming from her Thesis was published in the journal Marine Biodiversity (Galindo Estronza et al. 2016). Alexandra is now continuing her PhD research in the Schizas Lab on the commensal fauna of mesophotic reefs.
Galindo Estronza A, NV Schizas, M Alfaro (2016). Morphological and genetic species diversity in ostracods (Crustacea: Oligostraca) from Caribbean reefs. Marine Biodiversity DOI: 10.1007/s12526-016-0525-y
A pdf reprint has been submitted to SeaGrant with the Final Report.
2) Carlos Rivera Rosaly, carlos.rivera6@upr.edu, MS candidate in Marine Sciences. The theme of Rosaly's Thesis was the population genetics of commensal fauna associated with the mesophotic corals. Currently, Carlos is on leave from the Department.
3) Nick Hammerman, nick.hammerman@upr.edu, MS in Marine Sciences. The topic of Hammerman's Thesis was the population genomics of the mesophotic coral Agaricia lamarcki, which he successfully defended in December 2016. Nick Hammerman is continuing his PhD at the University of Queensland.
-List of Presentations
Schizas NV, Galindo-Estronza A, Alfaro M. Morphological and genetic species diversity in ostracods (Crustacea: Oligostraca) from Caribbean mesophotic reefs. 16th International Meiofauna Conference (IςIMCo), 3-8 July, 2016, Heraklion, Crete (poster)
Hammerman NM, Schizas NV, Alfaro M. Population Structure of the coral, Agaricia lamarcki from SW Puerto Rico and U.S. Virgin Islands. The 13th International Coral Reef Symposium (ICRS), 19-24 June, 2016, Honolulu, Hawaii (poster)
Garcia-Hernandez JE, Schizas NV, Alfaro ML, de Voogd NJ. Mesophotic Reef Sponges (Porifera) from Puerto Rico and the US Virgin Islands. The 13th International Coral Reef Symposium (ICRS), 19-24 June, 2016, Honolulu, Hawaii (talk)
Schizas NV. Introduction to Mesophotic reefs. CONGEN 2015. Workshop on Recent Advances in Conservation Genetics” January, 12 – 22, 2015, Rincon, Puerto Rico (Invited Talk)
Hammerman NM et al. Population connectivity of the coral Agaricia lamarcki from mesophotic and shallow reefs. 2015 Evolution Conference in Guaruja, Brazil, June 2015 (Poster).
Galindo-Estronza A., Alfaro M., Schizas NV. A morphological and molecular contribution on benthic ostracods with emphasis on the populations of the mesophotic reefs. ASLO, Grenada, Spain, 2015 (Poster)
List references for, books, chapters, and peer reviewed publications, in press, and submittals.
Published (All reprints available at http://www.schizaslab.com/)
Hammerman NM, RE Rivera-Vicens, MP Galaska, E Weil, RS Appledoorn, M Alfaro, NV Schizas (2017). Population connectivity of the plating coral, Agaricia lamarcki from southwest Puerto Rico. Coral Reefs DOI: 10.1007/s00338-017-1646-x
Lucas MQ, M Stat, M Smith, E Weil, NV Schizas (2016). Symbiodinium (internal transcribed spacer 2) diversity in the coral host Agaricia lamarcki (Cnidaria: Scleractinia) among shallow and mesophotic reefs in the Northern Caribbean (20–70 m). Marine Ecology doi: 10.1111/maec.12367
Corgosinho PHC, NV Schizas, M Alfaro (2016) A new species of Atergopedia (Copepoda: Harpacticoida: Novocriniidae) from a Caribbean mesophotic reef. Marine Biodiversity DOI 10.1007/s12526-016-0446-9
Galindo AM, NV Schizas, M Alfaro (2016). Morphological and genetic species diversity in ostracods (Crustacea: Oligostraca) from Caribbean reefs. Marine Biodiversity doi:10.1007/s12526-016-0525-y
Petrescu I, T Chatterjee, NV Schizas (2016). A new species of the genus Campylaspis (Crustacea: Cumacea) from the mesophotic reefs of St. John, US Virgin Islands. Caribbean Journal of Science 49:17-26
Guerra-Garcia JM, T Chatterjee, NV Schizas (2015). New genus and new species of Caprellidae (Crustacea: Peracarida: Amphipoda) from mesophotic coral ecosystems of Puerto Rico and St. Croix, Caribbean Sea. Zootaxa 4018 (1):080-096 http://dx.doi.org/10.11646/zootaxa.4018.1.4
Petrescu, I, T Chatterjee, NV Schizas (2016). New species and new records of Cumacea (Crustacea: Peracarida: Cumacea) from mesophotic reefs of Puerto Rico and US Virgin Islands, Caribbean Sea. Zootaxa 4199 (1): 001–078
Submitted Manuscripts:
Veglia AJ, NM Hammerman, CR Rivera, M Lucas, A Galindo Estronza, PH Corgosinho, and NV Schizas. Characterizing population structure of coralassociated fauna from mesophotic and shallow habitats in the Caribbean (In review). Journal of the Marine Biological Association of the United Kingdom.
Externally funded Travel Awards related to the proposal:
NSF-RAPID. The effects of Hurricane Maria on the mesophotic reefs of Puerto Rico. (2018). (PI – N Schizas; co-PIs Juan Cruz-Motta, Clark Sherman, Wilford Schmidt, Ernesto Weil)
MS student Alexandra Galindo-Estronza: Travel award from ASLO to attend the ASLO 2015 meeting, Grenada, Spain. Funded by ASLO.
MS student Nick Hammerman: Travel award from the Society of Systematic Biologists to attend the NSF-SSB Workshop in Model-based Molecular Systematics to take place on June 26, 2015 at the Evolution meeting in Guaruja, Brazil.
PI Nikolaos Schizas: Travel award from the Society of Systematic Biologists to attend the Evolution meeting in Guaruja, Brazil, June 26, 2015
First Mesophotic Reef Workshop – Biodiversity of Mesophotic Reefs took place on May 1, 2014 in the Biology Building (UPRM), where undergraduate/graduate students, professors and personnel of the Fundacion Surfiders, Rincon participated. The topics and schedule of the workshop were:
-Introductory Remarks - lecture Schizas/Alfaro
-Introduction to Mesophotic Reefs- lecture Schizas
-Macro- Meiofauna taxa of Mesophotic reefs- lecture Alexandra Galindo
-Morphology, phylogeny, diversity, of Copepods- lecture Paulo Corgosinho
May 2, 2014 (Biology Building) 9:00AM-12:00PM
-Morphology of selected Copepods –lab Paulo Corgosinho
-Microscopy work, dissections of Copepods – Lab Paulo Corgosinho
-Technical diving - Lecture Jaaziel Garcia
-Polychaetes of mesophotic reefs – Lecture Juan Laverde
The 2nd part of the workshop took place at Isla Magueyes on May, 2015. Schizas and Corgosinho showed the participants samples of the fauna associated with MCEs.
2nd and 3rd Mesophotic Workshop –in August 2015 and January 2016
A group of 15 undergraduate students in total participated in two mesophotic workshops during 2016 (eight students in the second workshop and seven in the 3rd). Students were guided through a well-designed introduction to how real taxonomic work is conducted. Some of the students were focused in the sorting of invertebrates from mesophotic samples collected during the 2910-2012 mesophotic cruises down to the Class taxonomic level. The rest of the students worked with the application of molecular techniques to measure biodiversity. The students went through all the procedures established to understand the high levels of diversity of invertebrates from mesophotic coral reefs to enhance our understanding of this ecosystem. Students started the protocol with the samples by washing samples over a 1 mm and a 0.125 mm sieve and all the material retained on the smaller sieve was stored in 100% ethanol. Organisms were hand sorted directly from the sediment or after a Ludox AM-30 colloidal silica resuspension and centrifugation step, used for mass-extraction of meiofauna and macrofauna (Nichols 1979) with the aid of a binocular microscope (Olympus SZH10) and stored in small plastic vials (1.5 mL) in the refrigerator for genetic and taxonomic work.
Students worked through one academic semester separating organisms to the lower taxonomic level possible. They were provided with guidelines and taxonomic identification keys to start learning the differences of external anatomy among the groups. In the molecular portion of the workshop, students learned all the protocols from DNA extraction to sequencing. Students macerated specimens preserved in 100% ethanol in a 1.5 mL centrifuge tube and extracted DNA using the DNeasy Blood & Tissue Kit (Qiagen Inc.) following the manufacturer’s guidelines.
Students checked for successful PCR reactions by loading 5l of the amplicon on a 1% TBE agarose gel. Electrophoresis was carried out for 45 min at 73 V. Amplified bands were visualized under UV light and captured digitally. Successful amplicons were sequenced in an ABI 3130XL Genetic Analyzer. DNA sequencing trace files were processed with Codon Code Aligner 4.1.1 and evolutionary analyses were conducted in
Mega v.5.1. We deposited the DNA sequences in GenBank and voucher specimens were placed in the Museum of Marine Invertebrates at Magueyes Island.
As a result of the mesophotic workshops, four students expressed interested in graduate studies at the Department of Marine Sciences and are either graduate students or applying to enter during the Fall semester of 2016.
-List PI’s supported
In addition to the students indicated above, we provide a breakdown of time and effort attributed to PI’s, Co-PI’s and associates (% time dedicated to project and amounts paid SG/Match during the report period, partial or full year) Personnel
Monica Alfaro (coPI)
Nikolaos V. Schizas