NOTE Garcia-Rodriguez 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 Seql-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 
Seql-16S 
16S 
12S-CR 
Seq2-16S 
Panulirus interruptus (California spiny lobster) 
Catalina Island, CA, USA 
33°26', 118°29' 
Mar 2003 
1 
Ensenada, B.C., Mexico 
31°50', 116°38' 
Aug 1999 
1 
3 
1 
Isla Guadalupe, B.C., Mexico 
29°00', 118°10' 
Dec 2002 
1 
3 
1 
Punta Eugenia, B.C.S., Mexico 
27°49', 115°06' 
Jun 1999 
2 
1 
Punta Abreojos, B.C.S., Mexico 
24°41', 113°34' 
Jun 1999 
2 
1 
San Juanico, B.C.S., Mexico 
24°14', 112°27' 
Oct 1999 
2 
1 
Bahia Magdalena, B.C.S., Mexico 
24°46', 112°06' 
Dec 1999 
2 
2 
1 
Panulirus inflatus (blue spiny lobster) 
Bahia Magdalena, B.C.S., Mexico 
24°46', 112°06' 
Nov 2001 
3 
3 
1 
21 
Mazatlan, Sin., Mexico 
23°13', 106°26' 
Aug 2002 
4 
3 
1 
14 
Las Penitas-Sayulita, Nay., Mexico 
21°00', 105°23' 
Aug 2002 
2 
3 
1 
24 
Barra de Navidad, Jal., Mexico 
19°12', 104°42' 
Jan 2005 
1 
3 
1 
15 
Zihuatanejo, Gue., Mexico 
17°37', 101°33' 
May 2005 
2 
1 
29 
Puerto Angel, Oax., Mexico 
15°39', 96°29' 
Nov 2002 
1 
23 
Panulirus gracilis (green spiny lobster) 
Bahia Magdalena, B.C.S., Mexico 
24°46', 112°06’ 
Nov 2001 
2 
Mazatlan, Sin., Mexico 
23°13’, 106°26' 
Aug 2002 
3 
4 
2 
21 
Las Penitas-Sayulita, Nay., Mexico 
21°00', 105°23' 
Aug 2002 
1 
5 
1 
23 
Punta Maldonado, Gue., Mexico 
16°20', 98°34' 
May 2005 
5 
2 
25 
Total 
20 
42 
18 
195 
PCR was performed in a total volume of 25 ,uL (Invitro- 
gene lx PCR buffer, 0.2 mM dNTP mix, 0.48 pM of each 
primer, 4.0 mM MgCl 2 , 1.25 U Taq DNA polymerase), 
with an iCycler thermal cycler (Bio-Rad Laboratories, 
Hercules, CA). The PCR program consisted of a dena- 
turation step at 94°C for 2 min, followed by 30 cycles 
of 1 min at 94°C, 1 min at 60°C, and 2 min at 72°C, 
followed by a final extension step of 4 min at 72°C. PCR 
products were confirmed using electrophoresis on 1% 
agarose gel, along with a molecular weight marker to 
estimate the fragment size. The gel was stained with 
SybrGold (Molecular Probes, Eugene, OR). All ampli- 
fied products were sequenced with primers 16Sar-L and 
16Sbr-H (Macrogen, Korea) and deposited in GenBank 
(accession numbers EF546597 through EF546616). 
The 16S rRNA gene sequences were aligned and ed- 
ited with the program Sequencher, vers. 4.5. (Gene 
Codes Corporation, Ann Harbor, MI). A neighbor-joining 
phylogram of haplotypes, based on the Kimura-2 param- 
eter model, was constructed in Molecular Evolutionary 
Genetics Analysis (MEGA) software vers. 3.0 (Kumar 
et al., 2004). Three sequences (one for each species) 
were used for the identification of diagnostic restriction 
enzyme recognition sites in ChromasPro software, vers. 
1.33 (Technelysium Pty Ltd, Tewantin, Queensland, 
Australia). Even though several restriction enzymes 
revealed various cut sites in the sequences, we selected 
only those that were easily seen on agarose gel. To 
check for consistency of the restriction enzyme cutting 
pattern in each species, the analysis was done with 17 
additional sequences. 
The RFLP pattern of the selected enzymes of another 
42 adult lobster specimens of the three species was 
tested for specimens collected at different locations 
(Table 1). Each digestion reaction occurred in a final 
7pL volume with 0.7 unit/pL of the selected enzyme, 
3.5 pL PCR product, and according to the manufactur- 
er’s instructions (New England Biolabs, Ipswich, MA). 
Reaction products for each enzyme were incubated for 
eight hours at the optimal temperature suggested by 
the manufacturer. For electrophoresis, restricted prod- 
ucts were run on 2% agarose gels and stained with 
SybrGold. A molecular weight marker was added to the 
agarose gel to estimate fragment size. To check con- 
sistency of intra- and interspecific nucleotide variation 
in addition to the 20 sequences, 195 sequences (a total 
