Mitochondrial DNA analysis as a tool for family and species identification of fish larvae: Emphasis on Snappers.

Mitochondrial DNA analysis as a tool for family and species identification of fish larvae: Emphasis on Snappers. Áurea E. Rodríguez, Juan C. Martínez-Cruzado, Ernesto Otero, Jorge R. García-Sais and Jennie T. Ramírez University of Puerto Rico Mayagüez Campus

Family: Lutjanidae (snappers) Commercially important Mainly tropical and subtropical marine 17 genera 103 species 4 Subfamilies: 1) Lutjaninae 2) Etelinae 3) Apsilinae 4) Paradicichthyinae

Subfamilies: 1. Lutjaninae» Lutjanus sp. (10)» Ocyurus chrysurus» Rhomboplites aurorubens 2. Etelinae:» Etelis oculatus» Pristipomoides macrophtalmus» Pristipomoides aquilonaris Lutjanus jocu (Dog snapper: Pargo perro o jocú) Lutjanus vivanus (Silk snapper: Chillo ojo amarillo ) 3. Apsilinae» Apsilus dentatus Etelis oculatus Queen snapper : Cartucho Apsilus dentatus (Black snapper: Chopa negra)

Problem Identification of snapper larvae to the species level is crucial for the understanding of their life cycle. Show very similar larvae but very different from adults. Existing identification keys based on morphometry are useless for most larvae (preflexion). 2.0 mm Pre - flexion larvae

Adult Yellowtail snapper 45 (cm/450mm) 5.0 mm Post-flexion 3.0 mm Larvae Unknown species Larvae Unknown species

Adult Yellowtail snapper 45 (cm/450mm) ~ 300X 5.0 mm Post-flexion 3.0 mm Larvae Unknown species Larvae Unknown species

Project Relevance Taxonomic identification of snapper larvae to the species level represents one of the main bottlenecks in our understanding of their early life cycle and dispersal patterns. Important to evaluate management options to restore the declining stocks of commercially exploited coral reef fishes, as for marine protected areas (MPAs).

Main Objective Identify snapper larvae [to species] by analysis of their mtdna 12S rrna gene sequence; comparing them to analog adult sequences

Collection 10 20 30 40 50.............................. Methods Cyprinus_carpio TACGAGCATT AGCTTAAAAC CCAAAGGACC TGACGGTGTC TCAGACCCCC Larva_(L107) TACGAGCATC AGCTTGAAAC CCAAAGGACT TGGCGGTGCT TTAGATCCAC Ocyurus_chysurus TACGAGCATC AGCTTGAAAC CCAAAGGACT TGGCGGTGCT TTAGATCCAC Larva(L108) TACGAGCATC AGCTTGAAAC CCAAAGGACT TGGCGGTGCT TTAGATCCAC Larva_(L111) TACGAGCATC AGCTTGAAAC CCAAAGGACT TGGCGGTGCT TTAGATCCAC Larva_(L21) TACGAGCATC AGCTTGAAAC CCAAAGGACT TGGCGGTGCT TTAGATCCAC Lutjanus_bucanella TACGAGCATC AGCTTGAAAC CCAAAGGACT TGGCGGTGCT TTAGATCCAC Lutjanus_vivanus TACGAGCATC AGCTTGAAAC CCAAAGGACT TGGCGGTGCT TTAGATCCAC Larva_(L20) TACGAGCATC AGCTTGAAAC CCAAAGGACT TGGCGGTGCT TTAGATCCAC Rhomboplites_aurorubens(66 TACGAGCATC AGCTTAAAAC CCAAAGGACT TGGCGGTGCT TTAGATCCAC Larva_(L24) TACGAGCATC AGCTTAAAAC CCAAAGGACT TGGCGGTGCT TTAGATCCAC Lutjanus_analis TACGAGCATT AGCTTGAAAC CCAAAGGACT TGGCGGTGCT TTAGATCCAC Larva_(L116) TACGAGCATT AGCTTGAAAC CCAAAGGACT TGGCGGTGCT TTAGATCCAC Lutjanus_mohogoni TACGAGCATC AGCTTGAAAC CCAAAGGACT TGGCGGTGCT TTAGATCCAC Lutjanus_synagris TACGAGCATC AGCTTGAAAC CCAAAGGACT TGGCGGTGCT TTAGATCCAC Larva_(L135) TACGAGCATC AGCTTGAAAC CCAAAGGACT TGGCGGTGCT TTAGATCCAC Lutjanus_griseus TACGAGCATC AGCTTGAAAC CCAAAGGACT TGGCGGTGCT TTAGATCCAC Lutjanus_jocu TACGAGCATC AGCTTAAAAC CCAAAGGACT TGGCGGTGCT TTAGATCCAC Lutjanus_apodus TACGAGCATC AGCTTAAAAC CCAAAGGACT TGGCGGTGCT TTAGATCCAC L136_7150 TACGAGCATC AGCTTAAAAC CCAAAGGACT TGGCGGTGCT TTAGATCCAC Adults Tissue from local catches Larvae Plankton Sampling sites: at La Parguera shelf edge El Hoyo La cuarta mella Preserved in 3% ethanol Lutjanus_cyanopterus TACGAGCATC AGCTTAAAAC CCAAAGGACT TGGCGGTGCT TTAGATCCAC Apsilus_dentatus TACGAGCATC AGCTTGAAAC CCAAAGGACT TGGCGGTGCT TTAGATCCAC Etelis_oculatus TACGAGCATT AGCTTAAAAC CCAAAGGACT TGGCGGTGCT TTAGATCCAC Pristipomoides_aquilonaris TACGAGCATT AGCTTGAAAC CCAAAGGACT TGGCGGTGCT TTAGACCCAC Standard measurements and photo documentation for each larvae

10 20 30 40 50.............................. Cyprinus_carpio TACGAGCATT AGCTTAAAAC CCAAAGGACC TGACGGTGTC TCAGACCCCC Methods Larva_(L107) TACGAGCATC AGCTTGAAAC CCAAAGGACT TGGCGGTGCT TTAGATCCAC Ocyurus_chysurus TACGAGCATC AGCTTGAAAC CCAAAGGACT TGGCGGTGCT TTAGATCCAC Larva(L108) TACGAGCATC AGCTTGAAAC CCAAAGGACT TGGCGGTGCT TTAGATCCAC Larva_(L111) TACGAGCATC AGCTTGAAAC CCAAAGGACT TGGCGGTGCT TTAGATCCAC Larva_(L21) TACGAGCATC AGCTTGAAAC CCAAAGGACT TGGCGGTGCT TTAGATCCAC Lutjanus_bucanella TACGAGCATC AGCTTGAAAC CCAAAGGACT TGGCGGTGCT TTAGATCCAC Lutjanus_vivanus TACGAGCATC AGCTTGAAAC CCAAAGGACT TGGCGGTGCT TTAGATCCAC Larva_(L20) TACGAGCATC AGCTTGAAAC CCAAAGGACT TGGCGGTGCT TTAGATCCAC Rhomboplites_aurorubens(66 TACGAGCATC AGCTTAAAAC CCAAAGGACT TGGCGGTGCT TTAGATCCAC Larva_(L24) TACGAGCATC AGCTTAAAAC CCAAAGGACT TGGCGGTGCT TTAGATCCAC Lutjanus_analis TACGAGCATT AGCTTGAAAC CCAAAGGACT TGGCGGTGCT TTAGATCCAC Larva_(L116) TACGAGCATT AGCTTGAAAC CCAAAGGACT TGGCGGTGCT TTAGATCCAC Lutjanus_mohogoni TACGAGCATC AGCTTGAAAC CCAAAGGACT TGGCGGTGCT TTAGATCCAC Lutjanus_synagris TACGAGCATC AGCTTGAAAC CCAAAGGACT TGGCGGTGCT TTAGATCCAC Larva_(L135) TACGAGCATC AGCTTGAAAC CCAAAGGACT TGGCGGTGCT TTAGATCCAC Lutjanus_griseus TACGAGCATC AGCTTGAAAC CCAAAGGACT TGGCGGTGCT TTAGATCCAC Lutjanus_jocu TACGAGCATC AGCTTAAAAC CCAAAGGACT TGGCGGTGCT TTAGATCCAC Lutjanus_apodus TACGAGCATC AGCTTAAAAC CCAAAGGACT TGGCGGTGCT TTAGATCCAC L136_7150 TACGAGCATC AGCTTAAAAC CCAAAGGACT TGGCGGTGCT TTAGATCCAC Lutjanus_cyanopterus TACGAGCATC AGCTTAAAAC CCAAAGGACT TGGCGGTGCT TTAGATCCAC Apsilus_dentatus TACGAGCATC AGCTTGAAAC CCAAAGGACT TGGCGGTGCT TTAGATCCAC Etelis_oculatus TACGAGCATT AGCTTAAAAC CCAAAGGACT TGGCGGTGCT TTAGATCCAC Pristipomoides_aquilonaris TACGAGCATT AGCTTGAAAC CCAAAGGACT TGGCGGTGCT TTAGACCCAC DNA Extraction (Qiagen Mini kit) Adults: muscle tissue Individual larvae PCR: mtdna 12S rrna gene ~ 450 bp Restriction fragment leght polymorphisms (RFLP s) : 11 enzymes Sequencing Sequence analysis: (MEGA 3.1) Alignment Tree construction

Results 4.8 mm Type 1 larvae 7.0 mm Type 2 larvae Two types of larvae morphologically characterized

Marker PBR-322 Control RFLP's Oc Oc Lg Lc Lb Lv Lap Lj ~ 450bp ~ 250bp ~ 200bp Restriction analysis with Mse I showing digestion patterns 0, 1 and 2.

RFLP's Enzymes Species Alu I Hinf I Mse I Haplotype Ocyurus chrysurus 2 1 1 A Lutjanus vivanus 1 1 0 B Lutjanus cyanopterus 2 0 2 Lutjanus jocu 2 0 2 C Lutjanus apodus 2 0 2 Lutjanus griseus 1 0 0 D Unknown larvae # 1 1 0 0 Lutjanus synagris 2 0 0 E Lutjanus analis 1 1 1 F Lutjanus bucanella 1 1 1 Unknown larvae # 2 1 0 2 G - Seven haplotypes (8 ind. per haplotype for most cases) - Four found in only one species and one larva

10 20 30 40 50.............................. Cyprinus_carpio TACGAGCATT AGCTTAAAAC CCAAAGGACC TGACGGTGTC TCAGACCCCC Larva_(L107) TACGAGCATC AGCTTGAAAC CCAAAGGACT TGGCGGTGCT TTAGATCCAC Ocyurus_chysurus TACGAGCATC AGCTTGAAAC CCAAAGGACT TGGCGGTGCT TTAGATCCAC Larva(L108) TACGAGCATC AGCTTGAAAC CCAAAGGACT TGGCGGTGCT TTAGATCCAC Larva_(L111) TACGAGCATC AGCTTGAAAC CCAAAGGACT TGGCGGTGCT TTAGATCCAC Larva_(L21) TACGAGCATC AGCTTGAAAC CCAAAGGACT TGGCGGTGCT TTAGATCCAC Lutjanus_bucanella TACGAGCATC AGCTTGAAAC CCAAAGGACT TGGCGGTGCT TTAGATCCAC Lutjanus_vivanus TACGAGCATC AGCTTGAAAC CCAAAGGACT TGGCGGTGCT TTAGATCCAC Larva_(L20) TACGAGCATC AGCTTGAAAC CCAAAGGACT TGGCGGTGCT TTAGATCCAC Rhomboplites_aurorubens(66 TACGAGCATC AGCTTAAAAC CCAAAGGACT TGGCGGTGCT TTAGATCCAC Larva_(L24) TACGAGCATC AGCTTAAAAC CCAAAGGACT TGGCGGTGCT TTAGATCCAC Lutjanus_analis TACGAGCATT AGCTTGAAAC CCAAAGGACT TGGCGGTGCT TTAGATCCAC Larva_(L116) TACGAGCATT AGCTTGAAAC CCAAAGGACT TGGCGGTGCT TTAGATCCAC Lutjanus_mohogoni TACGAGCATC AGCTTGAAAC CCAAAGGACT TGGCGGTGCT TTAGATCCAC Lutjanus_synagris TACGAGCATC AGCTTGAAAC CCAAAGGACT TGGCGGTGCT TTAGATCCAC Larva_(L135) TACGAGCATC AGCTTGAAAC CCAAAGGACT TGGCGGTGCT TTAGATCCAC Lutjanus_griseus TACGAGCATC AGCTTGAAAC CCAAAGGACT TGGCGGTGCT TTAGATCCAC Lutjanus_jocu TACGAGCATC AGCTTAAAAC CCAAAGGACT TGGCGGTGCT TTAGATCCAC Lutjanus_apodus TACGAGCATC AGCTTAAAAC CCAAAGGACT TGGCGGTGCT TTAGATCCAC L136_7150 TACGAGCATC AGCTTAAAAC CCAAAGGACT TGGCGGTGCT TTAGATCCAC Lutjanus_cyanopterus TACGAGCATC AGCTTAAAAC CCAAAGGACT TGGCGGTGCT TTAGATCCAC Apsilus_dentatus TACGAGCATC AGCTTGAAAC CCAAAGGACT TGGCGGTGCT TTAGATCCAC Etelis_oculatus TACGAGCATT AGCTTAAAAC CCAAAGGACT TGGCGGTGCT TTAGATCCAC Pristipomoides_aquilonaris TACGAGCATT AGCTTGAAAC CCAAAGGACT TGGCGGTGCT TTAGACCCAC 60 70 80 90 100.............................. Cyprinus_carpio CTAGAGGAGC CTGTTCTAGA ACCGATAACC CCCGTTCAAC CTCACCACTT Larva_(L107) CTAGAGGAGC CTGTTCTAGA ACCGATTACC CCCGTTCAAC CTCACCTTTC Ocyurus_chysurus CTAGAGGAGC CTGTTCTAGA ACCGATTACC CCCGTTCAAC CTCACCTTTC Larva(L108) CTAGAGGAGC CTGTTCTAGA ACCGATTACC CCCGTTCAAC CTCACCTTTC Larva_(L111) CTAGAGGAGC CTGTTCTAGA ACCGATTACC CCCGTTCAAC CTCACCTTTC Larva_(L21) CTAGAGGAGC CTGTTCTAGA ACCGATTACC CCCGTTCAAC CTCACCTTTC Lutjanus_bucanella CTAGAGGAGC CTGTTCTAGA ACCGATTACC CCCGTTCAAC CTCACCTTTC Lutjanus_vivanus CTAGAGGAGC CTGTTCTAGA ACCGATTACC CCCGTTCAAC CTCACCTTTC Larva_(L20) CTAGAGGAGC CTGTTCTAGA ACCGATTACC CCCGTTCAAC CTCACCTTTC Rhomboplites_aurorubens(66 CTAGAGGAGC CTGTTCTAGA ACCGATTACC CCCGTTCAAC CTCACCTTTC Larva_(L24) CTAGAGGAGC CTGTTCTAGA ACCGATTACC CCCGTTCAAC CTCACCTTTC Lutjanus_analis CTAGAGGAGC CTGTTCTAGA ACCGATTACC CCCGTTCAAC CTCACCTTTC Larva_(L116) CTAGAGGAGC CTGTTCTAGA ACCGATTACC CCCGTTCAAC CTCACCTTTC Lutjanus_mohogoni CTAGAGGAGC CTGTTCTAGA ACCGATTACC CCCGTTCAAC CTCACCTTTC Lutjanus_synagris CTAGAGGAGC CTGTTCTAGA ACCGATTACC CCCGTTCAAC CTCACCTTTC Larva_(L135) CTAGAGGAGC CTGTTCTAGA ACCGATTACC CCCGTTCAAC CTCACCTTTC Lutjanus_griseus CTAGAGGAGC CTGTTCTAGA ACCGATTACC CCCGTTCAAC CTCACCTTTC Lutjanus_jocu CTAGAGGAGC CTGTTCTAGA ACCGATTACC CCCGTTCAAC CTCACCTTTC Lutjanus_apodus CTAGAGGAGC CTGTTCTAGA ACCGATTACC CCCGTTCAAC CTCACCTTTC L136_7150 CTAGAGGAGC CTGTTCTAGA ACCGATTACC CCCGTTCAAC CTCACCTTTC Lutjanus_cyanopterus CTAGAGGAGC CTGTTCTAGA ACCGATTACC CCCGTTCAAC CTCACCTTTC Apsilus_dentatus CTAGAGGAGC CTGTTCTAGA ACCGATAACC CCCGTTCAAC CTCACCTTTC Etelis_oculatus CTAGAGGAGC CTGTTCTAGA ACCGATAACC CCCGTTCAAC CTCACCTTTT Pristipomoides_aquilonaris CTAGAGGAGC CTGTTCTAGA ACCGATAACC CCCGTTCAAC CTCACCTTTT 12S rrna gene sequence alignment

Phylogenetic relationships: Lutjanidae Family and Identified larvae 95 72 90 Lutjanus apodus L136 7150 Lutjanus jocu Lutjanus cyanopterus 66 Lutjanus synagris Larva (L135) 53 Lutjanus mohogoni 34 90 Lutjanus analis Larva (L116) Lutjanus griseus Bootstrap value 89 36 Lutjanus bucanella 28 Lutjanus vivanus 73 42 51 Larva (L107)? Ocyurus chysurus 100 Rhomboplites aurorubens(66) Larva (L24) Apsilus dentatus Etelis oculatus 85 Pristipomoides aquilonaris Cyprinus carpio 0.10 0.08 0.06 0.04 0.02 0.00 12S rrna gene Neighbor - Joining

Type 1 larvae (Lutjanids) Adult snapper (Ocyurus chysurus) Type 2 larvae identified as Serranus baldwini and Seranus tigrinus Serranus baldwini (adult) Serranus tigrinus (adult)

Species Identified Lutjanus apodus (Schoolmaster) Rhomboplites aurorubens (Vermillion snapper) Lutjanus synagris (Lane Snapper) Lutjanus analis (Mutton snapper) Ocyurus chrysurus? (Yellowtail) Serranus baldwini (Lantern bass) Serranus trigrinus (Harlequin bass)

Conclusion Mitochondrial DNA analysis of the 12S rrna gene is a reliable tool for family and species identification of fish larvae, as well as for phylogenetic reconstruction. This procedure is producing results that suggest that most larvae will be identified to the species level.

Benefits Information will be highly valuable to design more detailed research leading to characterize; dispersal, spawning and habitat selection for most of the species for the Lutjanidae family, that might be not accurately reported for the area. Knowledge gathered with this work leads to further research to characterize population structure and level of genetic connectivity of coral reef fishes populations within the Caribbean.

Acknowledgements My graduate committee: Dr. Juan C. Martínez, Dr. Ernesto Otero, Dr. Jorge (Reni) García and Dr. Fernando Bird. Luis (Puchito) Moreno; Parguera Fishing Village Manager, Nelson Crespo, Miguel (Gelo) Vargas, and Geño Piñeiro; Rincón Fishing Village. Roberto Ruiz, David Acosta, Mickey Amador, Miguel López (Pescadería Rosas), Iván López, and Froilán López for providing most adult tissue samples. To all fishermen, not only for the samples but also for sharing their priceless experiences. Mr. Marcos Rosado and Litzamar Irizarry for their invaluable help in field samplings. All staff at the Marine Science and Biology Departments. Laboratory partners at Marine Science and Biology Departments. Alliance for Graduate Education and the Professoriate (AGEP). Puerto Rico Sea Grant - NOAA award R-31-1-00.

Gracias!