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Mitochondrial DNA markers to identify 
commercial spiny lobster species ( Panulirus spp.) 
from the Pacific coast of Mexico: 
an application on phyllosoma larvae 
Francisco J. Garcia-Rodriguez 1 - 3 
German Ponce-Diaz 1 3 
Isabel Munoz-Garcia 2 
Rogelio Gonzalez-Armas 3 
Ricardo Perez-Enriquez 1 (contact author) 
Email address for R. Perez-Enriquez: rperez@cibnor.mx 
1 Centro de Investigaciones Biologicas del Noroeste (CIBNOR) 
Mar Bermeio 195, Col. Playa Palo de Santa Rita 
La Paz, Baha California Sur 23090, Mexico 
2 Facultad de Ciencias del Mar, Universidad Autonoma de Sinaloa 
Apdo. Postal 610 
Mazatlan, Sinaloa 82000, Mexico 
3 Centro Interdisciplinary de Ciencias Marinas-lnstituto Politecnico Nacional (CICIMAR-IPN) 
Apdo Postal 592 
La Paz, Baha California Sur 23000, Mexico 
Molecular markers based on mitochon- 
drial DNA (mtDNA) are extensively 
used to study genetic relationships. 
mtDNA has been used in phylogenetic 
studies to understand the evolutionary 
history of species because it is mater- 
nally inherited and is not subject to 
genetic recombination (Gyllensten et 
al., 1991). The high mutation rate of 
mtDNA makes it a useful tool for dif- 
ferentiating between closely related 
species (Brown et ah, 1979) — a tool 
that is especially important when 
significant variations occur between 
species, but not within species (Hill 
et al., 2001; Blair et al., 2006; Chow 
et al., 2006a). 
Species-level identification, based 
on molecular markers, can be very 
useful when morphological features 
alone do not provide sufficient dif- 
ferentiation or when only part of an 
organism is recovered. In fact, a few 
authors have successfully applied ge- 
netic markers for species identifica- 
tion based on remains recovered from 
fecal samples, parts of specimens, 
processed products, or larval and ju- 
venile forms (Chan et al., 2003; Pur- 
cell et al., 2004; Hsieh et ah, 2007). 
Three commercial lobster species 
inhabit the Pacific coast of Mexico: 
California spiny lobster ( Panulirus 
interruptus, west coast of California 
and the Baja California Peninsula); 
blue spiny lobster ( P . inflatus, south- 
ern Baja California Peninsula to the 
State of Oaxaca, Mexico); and green 
spiny lobster (P. gracilis, a tropical 
species from southern Baja California 
Peninsula to Peru) (Hendrickx, 1995). 
Taxonomic identification of adult lob- 
sters is easily done by morphological 
features (Hendrickx, 1995); however, 
alternative techniques are required 
for identification of larvae when mor- 
phological features are unable to pro- 
vide the means of identifying early 
life stages of spiny lobster species 
(Johnson, 1971; Munoz-Garcia et al., 
2004). Furthermore, discrimination 
between larvae of P. inflatus and P. 
gracilis could be especially difficult 
because of their overlapping distri- 
bution. 
The species-specific identification 
of larvae of other Panulirus species 
has also been difficult. Chow et al. 
(2006b) found intraspecific and in- 
tra-individual variation in appendage 
structures (the subexopodal spines) 
in the phyllosoma of P. ornatus (or- 
nate rock lobster) and P. versicolor 
(painted spiny lobster). Therefore, su- 
bexopodal spine arrangements may 
not be a useful diagnostic for distin- 
guishing between these two species. 
Because of these potential problems, 
several authors have suggested the 
use of molecular markers to identify 
spiny lobster larvae (Silberman and 
Walsh, 1992; Chow et al., 2006a, 
2006b; Konishi et al., 2006). 
In this study, the nucleotide varia- 
tions of the mitochondrial DNA (mtD- 
NA) in adult lobsters were investigat- 
ed to obtain genetic markers useful 
in identifying P. interruptus, P. in- 
flatus, and P. gracilis through either 
the polymerase chain reaction-restric- 
tion fragment length polymorphism 
(PCR-RFLP) analysis or species-spe- 
cific primers that amplify different 
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 
PCR-RFLP analysis 
DNA from adult specimens of the 
three species was taken from the 
following sites on the west coast of 
Mexico: P. interruptus from Baja Cali- 
fornia (n= 2) and Baja California Sur 
(n- 2); P. inflatus from Baja Califor- 
nia Sur (n = 3), Sinaloa (n- 4), Nayarit 
(n- 2), and Jalisco (n=l); and P. graci- 
lis from Baja California Sur (??= 2), 
Sinaloa (n = 3), and Nayarit (n=l) (see 
Table 1). 
A fragment of the 16S rRNA gene 
was amplified with primers 16Sar-L 
(5'-CGCCTGTTTATCAAAAACAT) 
and 16Sbr-H (5'-CCGGTCTGAACT- 
CAGATCACGT) (Palumbi, 1996). 
Manuscript submitted 7 June 2007. 
Manuscript accepted 11 December 2007. 
Fish. Bull. 106:204-212 (2008). 
The views and opinions expressed or 
implied in this article are those of the 
author and do not necessarily reflect 
the position of the National Marine 
Fisheries Service, NOAA. 
