Mitochondrial DNA analysis as a toolfor 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. RamírezUniversity of Puerto Rico

Mayagüez Campus

Family: Lutjanidae (snappers)

• Commercially important

• Mainly tropical and subtropical marine

• 17 genera 103 species• 4 Subfamilies:

1) Lutjaninae 2) Etelinae3) Apsilinae4) Paradicichthyinae

Subfamilies:1. Lutjaninae

» Lutjanus sp. (10)» Ocyurus chrysurus» Rhomboplites aurorubens

2. Etelinae:» Etelis oculatus» Pristipomoides macrophtalmus» Pristipomoides aquilonaris

3. Apsilinae» Apsilus dentatus

Lutjanus jocu (Dog snapper: Pargo perro o jocú)

Lutjanusvivanus

(Silk snapper: Chillo “ojo amarillo”)

Etelis oculatusQueen snapper : “Cartucho”

Apsilus dentatus(Black snapper: “Chopa negra)

Pre - flexion larvae

2.0 mm

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 (pre-flexion).

Adult – Yellowtail snapper

Larvae – Unknown species

3.0 mm

Larvae – Unknown species5.0 mm

Post-flexion

45 (cm/450mm)

Adult – Yellowtail snapper

Larvae – Unknown species

3.0 mm

Larvae – Unknown species5.0 mm

Post-flexion

45 (cm/450mm)

~ 300X

Project RelevanceTaxonomic 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).

Identify snapper larvae [to species] by analysis of their

mtDNA 12S rRNA gene sequence; comparing them to

analogadult sequences

Main Objective

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

CollectionAdults – Tissue from local catchesLarvae – Plankton

Sampling sites: at La Parguera shelf edge

“El Hoyo”“La cuarta mella”

Preserved in 3% ethanol• Standard measurements and photo

documentation for each larvae

Methods

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

• 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

Methods

Two types of larvae morphologically characterized

Type 1 larvae

4.8 mm

Type 2 larvae

7.0 mm

Results

Restriction analysis with Mse I showing digestion patterns 0, 1 and 2.

RFLP'sOc Oc Lg Lc Lb Lv Lap LjC

ontr

ol

Mar

ker

PBR

-322

~ 450bp

~ 250bp

~ 200bp

RFLP's

- Four found in only one species and one larva

EnzymesSpecies Alu I Hinf I Mse I HaplotypeOcyurus chrysurus 2 1 1 ALutjanus vivanus 1 1 0 BLutjanus cyanopterus 2 0 2Lutjanus jocu 2 0 2 CLutjanus apodus 2 0 2Lutjanus griseus 1 0 0 DUnknown larvae # 1 1 0 0Lutjanus synagris 2 0 0 ELutjanus analis 1 1 1 FLutjanus bucanella 1 1 1Unknown larvae # 2 1 0 2 G

- Seven haplotypes (8 ind. per haplotype for most cases)

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

12S rRNA gene Neighbor - Joining

Lutjanus apodus

L136 7150

Lutjanus jocu

Lutjanus cyanopterus

Lutjanus synagris

Larva (L135)

Lutjanus mohogoni

Lutjanus analis

Larva (L116)

Lutjanus griseus

Lutjanus bucanella

Lutjanus vivanus

Larva (L107)

Ocyurus chysurus

Rhomboplites aurorubens(66)

Larva (L24)

Apsilus dentatus

Etelis oculatus

Pristipomoides aquilonaris

Cyprinus carpio

85

100

90

72

73

95

90

89

66

34

53

36

28

51

42

0.000.020.040.060.080.10

Bootstrap value

?

Type 1 larvae (Lutjanids)

Type 2 larvae identified as Serranus baldwini and Seranus tigrinus

Adult snapper (Ocyurus chysurus)

Serranus baldwini (adult) Serranus tigrinus (adult)

Species IdentifiedLutjanus 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)

ConclusionMitochondrial DNA analysis of the 12S rRNAgene 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 (PescaderíaRosas), 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!