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Fishery Bulletin 106(2) 
of 215) from different geographical sites of P. inflatus 
(n- 126) and P. gracilis (n = 69) were analyzed. This 
extension was important because the larvae of these 
species are found in the same area and there is a higher 
possibility that misidentification will occur when using 
only morphological criteria (Table 1). 
Panulirus penicillatus (pronghorn spiny lobster) is 
found along the Pacific coast of Mexico and even though 
it is found in small numbers, it could be confused with 
the larvae of the other species. The restriction pattern 
of a 16S rRNA gene sequence reported in the GenBank 
(accession number AF337974) was compared to those of 
the other three lobster species. 
Multiplex PCR analysis 
Species-specific primers for P. interruptus have been 
described in a previous study (Garcia-Rodriguez and 
Perez-Enriquez, 2006; GenBank accession number 
EF565146). Based on this sequence, a reverse primer (5'- 
TGGTGTGATCCCGTTACTTG) was designed to amplify 
a -1250 base-pair (bp) mtDNA fragment containing the 
12S rRNA gene and the control region in P. inflatus and 
P. gracilis by means of Weider et al.’s (1996) forward 
primer (srRNA: 5 -CAGGGTATCTAATCCTGGTT). PCR 
thermal cycling consisted of an initial denaturation 
of 2 min at 94°C, 35 cycles of 1 min at 94°C, 1 min at 
58°C (for P. inflatus, but 52°C for P. gracilis), 2 min at 
72°C, and a final cycle of 4 min at 72°C. PCR products 
from adult specimens of each species were sequenced by 
using the srRNA primer. The Primer3 program (Rozen 
and Skaletsky, 2000) was used to design specific prim- 
ers for each species; these primers would have similar 
melting temperatures but products of different sizes 
(Fig. 1). Sequences obtained with specific primers were 
deposited in GenBank (accession numbers EF565144 
and EF565145). 
The specificity and reliability of the multiplex PCR 
reaction with 18 previously identified adult lobsters 
collected in different regions were tested. For P. inter- 
ruptus ( n-1 ), specimens were obtained from California, 
Isla Guadalupe, Baja California, and Baja California 
Sur; for P. inflatus (n- 6), specimens were collected from 
Baja California Sur, Sinaloa, Nayarit, Jalisco, Guerrero, 
and Oaxaca; for P. gracilis, specimens (n = 5) came from 
Sinaloa, Nayarit, and Guerrero (Table 1). 
Multiplex PCR reactions were carried out in a total 
volume of 12.5 pL, mixing 0.48 pM of each primer (one 
common primer: srRNA, and the three species-spe- 
cific primers: LanCR-R ( 5 -AAAAATTCAGGCTAAT- 
GGA), PinRCl-b (5'-GATGGCCCATTACCGAACTA), 
and PgraRCl-b (5 -TTGTGAAACGTCTGTTTACATT- 
TATTT)), Invitrogene lx PCR buffer, 0.2 mM dNTP 
mix, 4.0 mM MgCl 2 , and 0.625 U Taq DNA polymerase. 
PCR thermal cycling consisted of an initial denatur- 
ation of 2 min at 94°C, followed by 30 cycles of 1 min 
at 94°C, 1 min at 59°C, 2 min at 72°C, and a final cycle 
of 4 min at 72°C. The amplified products from each 
species were distinguished by electrophoresis on 1.0 % 
agarose gels. 
Application of molecular markers 
Phyllosoma larvae were collected during an oceano- 
graphic cruise outside the Gulf of California in November 
2004. Plankton sampling consisted of horizontal surface 
tows of a neuston collection net at 3.5 knots (6.4 km/h) 
for 5 min (see Gonzalez-Armas et ah, 1999). Sampling 
gear consisted of a rectangular plankton net of Nytex 
with 505-pm mesh; it had a 30x50 cm mouth area and 
the net was 3 m long. Phyllosoma larvae were sorted by 
hand after each tow and fixed with 70% ethanol. 
A fragment of the pereiopods, antenna, or eyes was 
obtained from each lobster larva for DNA isolation by 
lysing the tissue in 15 uL lysis buffer (10 mM Tris- 
HCL, pH 8.3, 50 mM KC1, 0.5% Tween-20), and 1.87 
pL (4 pg/pL) proteinase K and incubated overnight at 
55°C. Each reaction was then maintained at 95°C for 
approximately 10 min and stored at 4°C until analy- 
sis. Steps of PCR-RFLP and PCR multiplex for larval 
lobsters were the same as those carried out for adult 
specimens. 
Before genetic analysis, 46 lobster larvae collected 
from the Pacific Ocean were categorized into four pos- 
sible groups based on morphological criteria (Johnson 
and Knight, 1966; Johnson, 1971; Baez, 1983): group 1) 
Panulirus inflatus- like (n=17); group 2) Panulirus graci- 
Zis-like {n = 8); group 3) Panulirus inflatus-gracilis-like 
(n =12); and group 4) Panulirus- like (n= 9). 
Results 
PCR-RFLPs 
The 16S rRNA gene fragment was correctly amplified 
in the three lobster species. The size of the 16S rRNA 
gene fragment amplified in the three spiny lobster spe- 
cies was estimated at 563 bp and did not differ in size 
among species. There was wide inter- and intraspecific 
variation in nucleotide sequences among the lobster spe- 
cies, indicating the potential for species discrimination. 
A haplotype tree showed the aggregation of haplotypes 
according to each species (Fig. 2). 
From the analysis of 20 sequences, two restriction 
enzymes (BsmAl, GTCTCN' and Hinfl, GANTC) were 
selected that allowed discrimination among the three 
species (Fig. 3). The restriction products of the BsmAl 
digests were two fragments (-401 and -162 bp) in both 
P. interruptus and P. inflatus (named “haplotype A”) 
and three fragments (-401, -115, and -47 bp) in P. 
gracilis (named “haplotype B”). Restriction digests 
that used Hinfl produced two fragments (-440 and - 
123 bp) for P. interruptus and most P. gracilis speci- 
mens (named “haplotype A”), but did not cut P. infla- 
tus (named “haplotype B”). One P. gracilis sequence 
appeared to be haplotype B due to the absence of the 
Hinfl site. The composite haplotypes were constructed 
by the combination of the haplotype names of each 
enzyme, resulting in AA for P. interruptus, AB for P. 
inflatus, and either BA (98.7%) or BB (1.3%) for P. grac- 
