204 Mitochondrial DNA markers to identify structures (the subexopodal spines) in the phyllosoma of P. ornatus (or- commercial spiny lobster species (Panulirus spp.) nate rock lobster) and P. versicolor (painted spiny lobster). Therefore, su- from the Pacific coast of Mexico: bexopodal spine arrangements may an application on phyllosoma larvae not be a useful diagnostic for distin- guishing between these two species. Francisco J. García-Rodríguez1,3 Because of these potential problems, several authors have suggested the Germán Ponce-Díaz1,3 use of molecular markers to identify Isabel Muñoz-García2 spiny lobster larvae (Silberman and Rogelio González-Armas3 Walsh, 1992; Chow et al., 2006a, Ricardo Perez-Enriquez1 (contact author) 2006b; Konishi et al., 2006). In this study, the nucleotide varia- Email address for R. Perez-Enriquez: [email protected] tions of the mitochondrial DNA (mtD- 1 Centro de Investigaciones Biológicas del Noroeste (CIBNOR) NA) in adult lobsters were investigat- Mar Bermejo 195, Col. Playa Palo de Santa Rita ed to obtain genetic markers useful La Paz, Baha California Sur 23090, Mexico in identifying P. interruptus, P. in- 2 Facultad de Ciencias del Mar, Universidad Autónoma de Sinaloa flatus, and P. gracilis through either Apdo. Postal 610 the polymerase chain reaction-restric- Mazatlán, Sinaloa 82000, Mexico tion fragment length polymorphism 3 Centro Interdisciplinario de Ciencias Marinas-Instituto Politécnico Nacional (CICIMAR-IPN) (PCR-RFLP) analysis or species-spe- Apdo. Postal 592 cific primers that amplify different La Paz, Baha California Sur 23000, Mexico size fragments in a multiplex reac- tion. These techniques were used to identify phyllosoma larvae col- lected outside the Gulf of California. Materials and methods Molecular markers based on mitochon- Three commercial lobster species drial DNA (mtDNA) are extensively inhabit the Pacific coast of Mexico: PCR-RFLP analysis used to study genetic relationships. California spiny lobster (Panulirus mtDNA has been used in phylogenetic interruptus, west coast of California DNA from adult specimens of the studies to understand the evolutionary and the Baja California Peninsula); three species was taken from the history of species because it is mater- blue spiny lobster (P. inflatus, south- following sites on the west coast of nally inherited and is not subject to ern Baja California Peninsula to the Mexico: P. interruptus from Baja Cali- genetic recombination (Gyllensten et State of Oaxaca, Mexico); and green fornia (n=2) and Baja California Sur al., 1991). The high mutation rate of spiny lobster (P. gracilis, a tropical (n=2); P. inflatus from Baja Califor- mtDNA makes it a useful tool for dif- species from southern Baja California nia Sur (n=3), Sinaloa (n=4), Nayarit ferentiating between closely related Peninsula to Peru) (Hendrickx, 1995). (n=2), and Jalisco (n=1); and P. graci- species (Brown et al., 1979)—a tool Taxonomic identification of adult lob- lis from Baja California Sur (n=2), that is especially important when sters is easily done by morphological Sinaloa (n=3), and Nayarit (n=1) (see significant variations occur between features (Hendrickx, 1995); however, Table 1). species, but not within species (Hill alternative techniques are required A fragment of the 16S rRNA gene et al., 2001; Blair et al., 2006; Chow for identification of larvae when mor- was amplified with primers 16Sar-L et al., 2006a). phological features are unable to pro- (5ʹ-CGCCTGTTTATCAAAAACAT) Species-level identification, based vide the means of identifying early and 16Sbr-H (5ʹ-CCGGTCTGAACT- on molecular markers, can be very life stages of spiny lobster species CAGATCACGT) (Palumbi, 1996). useful when morphological features (Johnson, 1971; Muñoz-García et al., alone do not provide sufficient dif- 2004). Furthermore, discrimination ferentiation or when only part of an between larvae of P. inflatus and P. organism is recovered. In fact, a few gracilis could be especially difficult Manuscript submitted 7 June 2007. authors have successfully applied ge- because of their overlapping distri- Manuscript accepted 11 December 2007. netic markers for species identifica- bution. Fish. Bull. 106:204–212 (2008). tion based on remains recovered from The species-specific identification fecal samples, parts of specimens, of larvae of other Panulirus species The views and opinions expressed or implied in this article are those of the processed products, or larval and ju- has also been difficult. Chow et al. author and do not necessarily reflect venile forms (Chan et al., 2003; Pur- (2006b) found intraspecific and in- the position of the National Marine cell et al., 2004; Hsieh et al., 2007). tra-individual variation in appendage Fisheries Service, NOAA. NOTE García-Rodríguez et al.: Mitochondrial DNA markers to identify Panulirus spp. 205 Table 1 Sampling sites and dates for each lobster species along the Pacific coast of California and Mexico. The number of samples in the Seq1-16S column refers to the sequences used to find diagnostic restriction sites to discriminate lobster species; RFLP and Multiplex columns are the number of larvae analyzed by the RFLP and multiplex analysis, respectively; Seq2-16S column are the sequences used to evaluate consistency of restriction patterns. Number of samples Sampling RFLP Multiplex Sampling sites Location date Seq1-16S 16S 12S-CR Seq2-16S Panulirus interruptus (California spiny lobster) Catalina Island, CA, USA 33°26ʹ, 118°29ʹ Mar 2003 1 Ensenada, B.C., Mexico 31°50ʹ, 116°38ʹ Aug 1999 1 3 1 Isla Guadalupe, B.C., Mexico 29°00ʹ, 118°10ʹ Dec 2002 1 3 1 Punta Eugenia, B.C.S., Mexico 27°49ʹ, 115°06ʹ Jun 1999 2 1 Punta Abreojos, B.C.S., Mexico 24°41ʹ, 113°34ʹ Jun 1999 2 1 San Juanico, B.C.S., Mexico 24°14ʹ, 112°27ʹ Oct 1999 2 1 Bahía Magdalena, B.C.S., Mexico 24°46ʹ, 112°06ʹ Dec 1999 2 2 1 Panulirus inflatus (blue spiny lobster) Bahía Magdalena, B.C.S., Mexico 24°46ʹ, 112°06ʹ Nov 2001 3 3 1 21 Mazatlán, Sin., Mexico 23°13ʹ, 106°26ʹ Aug 2002 4 3 1 14 Las Peñitas-Sayulita, Nay., Mexico 21°00ʹ, 105°23ʹ Aug 2002 2 3 1 24 Barra de Navidad, Jal., Mexico 19°12ʹ, 104°42ʹ Jan 2005 1 3 1 15 Zihuatanejo, Gue., Mexico 17°37ʹ, 101°33ʹ May 2005 2 1 29 Puerto Angel, Oax., Mexico 15°39ʹ, 96°29ʹ Nov 2002 1 23 Panulirus gracilis (green spiny lobster) Bahía Magdalena, B.C.S., Mexico 24°46ʹ, 112°06ʹ Nov 2001 2 Mazatlán, Sin., Mexico 23°13ʹ, 106°26ʹ Aug 2002 3 4 2 21 Las Peñitas-Sayulita, Nay., Mexico 21°00ʹ, 105°23ʹ Aug 2002 1 5 1 23 Punta Maldonado, Gue., Mexico 16°20ʹ, 98°34ʹ May 2005 5 2 25 Total 20 42 18 195 PCR was performed in a total volume of 25 μL (Invitro- enzyme recognition sites in ChromasPro software, vers. gene 1× PCR buffer, 0.2 mM dNTP mix, 0.48 μM of each 1.33 (Technelysium Pty Ltd, Tewantin, Queensland, primer, 4.0 mM MgCl2, 1.25 U Taq DNA polymerase), Australia). Even though several restriction enzymes with an iCycler thermal cycler (Bio-Rad Laboratories, revealed various cut sites in the sequences, we selected Hercules, CA). The PCR program consisted of a dena- only those that were easily seen on agarose gel. To turation step at 94°C for 2 min, followed by 30 cycles check for consistency of the restriction enzyme cutting of 1 min at 94°C, 1 min at 60°C, and 2 min at 72°C, pattern in each species, the analysis was done with 17 followed by a final extension step of 4 min at 72°C. PCR additional sequences. products were confirmed using electrophoresis on 1% The RFLP pattern of the selected enzymes of another agarose gel, along with a molecular weight marker to 42 adult lobster specimens of the three species was estimate the fragment size. The gel was stained with tested for specimens collected at different locations SybrGold (Molecular Probes, Eugene, OR). All ampli- (Table 1). Each digestion reaction occurred in a final fied products were sequenced with primers 16Sar-L and 7μL volume with 0.7 unit/μL of the selected enzyme, 16Sbr-H (Macrogen, Korea) and deposited in GenBank 3.5 μL PCR product, and according to the manufactur- (accession numbers EF546597 through EF546616). er’s instructions (New England Biolabs, Ipswich, MA). The 16S rRNA gene sequences were aligned and ed- Reaction products for each enzyme were incubated for ited with the program Sequencher, vers. 4.5. (Gene eight hours at the optimal temperature suggested by Codes Corporation, Ann Harbor, MI). A neighbor-joining the manufacturer. For electrophoresis, restricted prod- phylogram of haplotypes, based on the Kimura-2 param- ucts were run on 2% agarose gels and stained with eter model, was constructed in Molecular Evolutionary SybrGold. A molecular weight marker was added to the Genetics Analysis (MEGA) software vers. 3.0 (Kumar agarose gel to estimate fragment size. To check con- et al., 2004). Three sequences (one for each species) sistency of intra- and interspecific nucleotide variation were used for the identification of diagnostic restriction in addition to the 20 sequences, 195 sequences (a total 206 Fishery Bulletin 106(2) of 215) from different geographical sites of P. inflatus Application of molecular markers (n=126) and P. gracilis (n=69) were analyzed. This extension was important because the larvae of these Phyllosoma larvae were collected during an oceano- species are found in the same area and there is a higher graphic cruise outside the Gulf of California in November possibility that misidentification will occur when using 2004. Plankton sampling consisted of horizontal surface only morphological criteria (Table 1). tows of a neuston collection net at 3.5 knots (6.4 km/h) Panulirus penicillatus (pronghorn spiny lobster) is for 5 min (see González-Armas et al., 1999).