RESEARCH PAPER

Mitochondrial haplotype variation in wild trout populations(Teleostei: Salmonidae) from northwestern Mexico

Faustino Camarena-Rosales Æ Gorgonio Ruiz-Campos Æ Jorge De La Rosa-Velez ÆRichard L. Mayden Æ Dean A. Hendrickson Æ Alejandro Varela-Romero ÆFrancisco J. Garcıa de Leon

Received: 19 September 2005 / Accepted: 13 March 2007 / Published online: 3 May 2007

� Springer Science+Business Media B.V. 2007

Abstract The variation and composition of Mexi-

can wild trout mitochondrial DNA haplotypes

throughout northwestern Mexico was determined by

means of polymerase chain reaction–restriction frag-

ment polymorphism analysis (PCR–RFLP), of one

region of mitochondrial DNA between cytochrome b

and the D-loop. This analysis was based on 261

specimens taken in 12 basins and four hatcheries

from northwestern Mexico. From 23 haplotypes, 15

wild trout haplotypes were identified and classified in

four groups: (1) one restricted to Nelson’s trout

(Oncorhynchus mykiss nelsoni), (2) four restricted to

Rıo Mayo and RıoYaqui trout (O. mykiss sspp.), (3)

six to Mexican golden trout (O. chrysogaster) with

two subgroups, and (4) one exclusive to Rıo Piaxtla

trout. Distributions of native haplotypes broadly

overlap the distribution of non-native hatchery rain-

bow trout reflecting the historical management of

introductions of exotic rainbow trout and the artificial

transference of these trout among basins.

Keywords Mitochondrial DNA � PCR–RFLP �Wild trout � Oncorhynchus � Mexico

Introduction

Wild trout from northwestern Mexico are considered

to represent two nominal species, the coastal rainbow

F. Camarena-Rosales � G. Ruiz-Campos (&)

Facultad de Ciencias, Universidad Autonoma de Baja

California, Apdo. Postal 233, Ensenada, Baja California

22800, Mexico

e-mail: gruiz@uabc.mx

J. De La Rosa-Velez

Facultad de Ciencias Marinas,

Universidad Autonoma de Baja California,

Apdo. Postal 653, Ensenada, Baja California 22800,

Mexico

R. L. Mayden

Department of Biology, Saint Louis University, St. Louis,

MO, USA

D. A. Hendrickson

Texas Memorial Museum, Texas Natural History

Collections, University of Texas, Austin, TX, USA

A. Varela-Romero

Departamento de Investigaciones Cientıficas y

Tecnologicas de la Universidad de Sonora, Apdo. Postal

1819, Hermosillo, Sonora 83000, Mexico

F. J. Garcıa de Leon

Centro de Investigaciones Biologicas del Noroeste, S.C.

Programa Planeacion Ambiental y Conservacion, Apdo.

Postal 128, La Paz, Baja California Sur 23000, Mexico

G. Ruiz-Campos

PMB 064, P.O. Box 189003-064, Coronado, CA 92178, USA

123

Rev Fish Biol Fisheries (2008) 18:33–45

DOI 10.1007/s11160-007-9060-z

trout or Nelson’s trout, Oncorhynchus mykiss nelsoni,

endemic to the Sierra San Pedro Martir (SSPM), Baja

California (Evermann 1908; Nelson 1921; Snyder

1926; Smith 1991; Ruiz-Campos and Pister 1995),

and the Mexican golden trout O. chrysogaster of the

Sierra Madre Occidental (SMO) (Needham and Gard

1959; Behnke 2002; Hendrickson et al. 2003; Ruiz-

Campos et al. 2003) (Fig. 1).

The natural distribution of Nelson’s trout extended

throughout a 24-km segment of the Rıo Santo

Domingo (also referenced as San Antonio de Muril-

los or San Ramon) between Rancho San Antonio and

a high waterfall that blocked further upstream

movement of trout (Evermann 1908; Snyder 1926;

Ruiz-Campos and Pister 1995). Between 1929 and

1941 this trout was introduced into other tributaries of

the Rıo Santo Domingo (La Mision, La Grulla, La

Zanja and El Potrero) as well as to the Rıo San

Rafael, increasing its distribution in the SSPM (Ruiz-

Campos and Pister 1995).

The Mexican golden trout is endemic to headwater

tributaries of the Rıo Fuerte, Rıo Sinaloa and Rıo

Culiacan drainages in the SMO (Needham and Gard

1959, 1964; Miller 1950; Behnke 1991, 2002;

Hendrickson et al. 2003; Ruiz-Campos et al. 2003).

Several undescribed populations of native trout found

both north and south of the Mexican golden trout

(Behnke 1991; Hendrickson et al. 2003; Ruiz-Campos

et al. 2003; Mayden 2004) have been referred to as

evolutionary species (sensu Mayden 2004). The north-

ern forms include the Rıo Yaqui and Rıo Mayo trout;

while the southern undescribed forms apparently exist in

the rıos San Lorenzo, Piaxtla, Presidio, Baluarte and

Acaponeta (Ruiz-Campos et al. 2003). Recent mito-

chondrial DNA and microsatellites analyses have found

several unique genetic characters in the Rıo Yaqui trout

(Nielsen et al. 1997; Nielsen and Sage 2001).

One of the primary factors that threaten the genetic

integrity of the Mexican native trout in the SMO, is

the establishment of non-native hatchery strains of

rainbow trout. Hatcheries housing non-native stocks

in facilities ranging from rustic ponds to raceway

systems with breeding and rearing areas and are

operating on tributaries of almost all drainage

systems that have native Mexican trout (Hendrickson

et al. 2003; Ruiz-Campos et al. 2003; this work).

Unfortunately, the presence of native trout in the

SMO and the potential threats inherent in non-native

introductions have been ignored by the Mexican

governmental agencies during their programs support-

ing rural aquaculture. The establishment of hatcheries

for exotic rainbow trout in the same drainages as native

trout have resulted in the escape of cultured individuals

into adjacent streams (Ruiz-Campos et al. 2003). This

Fig. 1 Sampling field localities of wild trout from northwest-

ern Mexico. Abbreviation: SR1 = San Rafael; SD1 = La Grulla;

SD2 = San Antonio (Baja California) ; Y1 = San Antonio

(Sonora); Y2 = Los Pescados; Y3 = La Presita; M1 = El

Concheno; M2 = Potrero de Gil; F1 = La Onza; F2 = Arroyo

Verde; S1 = Casa Quemada; C1 = Mesa San Rafael; SL1 = La

Sidra; P1 = La Quebrada; A1 = Los Metates; B1 = Coscomate.

Hatcheries in black box: H1 = San Antonio, H2 = Potrero de

Gil, H3 = Vencedores and H4 = Los Metates

34 Rev Fish Biol Fisheries (2008) 18:33–45

123

event will result in hybridization and will increase the

genetic variation within the recipient population by

increasing the number of genetics forms contained

within that population. Until a few years ago no

regulations for the capture, use and transplant of trout

had been established in Mexico. However with the

implementation of the Mexican Official Norm in 1994

(SEDESOL 1994), Nelson’s trout, and later the Mexican

golden trout (SEMARNAT 2002) were provided federal

protection; the remaining are still unprotected.

In order to determine the current genetic and

population status of native trout from northwestern

Mexico, we collected trout specimens from diverse

localities in 12 hydrologic basins in the States of Baja

California, Sonora, Chihuahua and Durango during

six expeditions (October 2000–September 2001). The

objectives of the present study were: (1) to detect genetic

markers using PCR–RFLP for the differentiation of

populations of wild trout, (2) to determine markers

obtained using PCR–RFLP that might allow discrimi-

nation of wild trout and introduced forms, and (3) to

document current distributions of mtDNA haplotypes.

Study area

The study area is part of two major hydrological regions

of northwestern Mexico (Tamayo and West 1964): the

Baja California Drainage represented by small streams

that rise on the western slope of the SSPM and drain to

the Pacific Ocean, and the Northern Pacific Drainage

represented by large rivers that rise on the western slope

of the SMO, cross the coastal lowlands from Sonora to

central Nayarit, and finally empty into the Gulf of

California (Fig. 1). Biogeographically, this region

belongs to SMO province and the Mountain Meso-

america region, which is a transition zone between the

Holarctic and Neotropical kingdoms (Rzedowski 1986).

A more detailed description of the study area and

collecting sites is found in Hendrickson et al. (2003) and

Ruiz-Campos et al. (2003).

Methods

Trout sampling

Trout specimens were sampled from October 2000 to

September 2001 in 17 streams found in 12 basins

(Fig. 1). Sampling sites are located in elevations

ranging from 560 m (at SSPM) to 2,560 m above sea

level (at SMO). The geographic location of each

sample site was determined using GPS (Table 1).

Stream names were taken from topographic maps

(1:250,000) published by the Instituto Nacional de

Estadıstica Geografıa e Informatica (INEGI).

Trout were captured along 200 m transects in each

stream using AC Smith-RootTM model 15-B POW

electrofishing equipment. Hook and line was also used

in localities where electrofishing was difficult (La Grulla

and El Concheno). Since most sampled populations

appeared to be at low densities and any mortality could

have negative impacts to the population, sample size

was limited to 10–15 individuals per locality (except at

El Concheno where n = 3). Samples of non-native

rainbow trout were also obtained from four hatcheries

located in the same river basins, generally near locations

where wild trout were collected. In the field, freshly

captured specimens were individually labeled, placed in

plastic bags, and placed on dry ice for transportation to

the laboratory where they were stored at�808C.

PCR–RFLP

Total DNA extractions were performed using a

phenol–chloroform protocol (Sambrook et al. 1989).

In the PCR technique we used specific primers for

trout mitochondrial DNA (mtDNA) (Bernatchez and

Danzmann 1993; Bernatchez and Osinov 1995) to

amplify the region between cytochrome b (50-CTTGAAAAACCACCGTTGTTA-30) and the D-

loop (50-GTGTTATGCTTTAGTTAAGC-30). These

primers have reported in other studies (Imsiridou

et al. 2003) and will be used here to construct the

hypothetical pattern of fragment to compare with the

electrophoretical pattern of bands. Amplification

products were approximately 2354 bp in length and

included complete cytochrome b and the D-Loop.

The mixture reaction contained 20–100 ng of DNA,

150 ng of each primer, 200 mM of each dNTP, 1 ml of

Taq polymerase and the corresponding buffer,

1.65 mM MgCl2 and H2O in a final volume of

50 ml. Amplification was carried out using a Perkin

Elmer thermal cycler (model 480) with the following

conditions: initial denaturation at 948C for 5 min,

followed by 35 cycles at 948C for 30 s, 528C for

1 min, 728C for 2.30 min, and finally one extension of

728C for 10 min. The products were visualized using

ethidium bromide in 0.8% agarose gels in Tris Borato

buffer (TBE) of electrophoresis.

Rev Fish Biol Fisheries (2008) 18:33–45 35

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36 Rev Fish Biol Fisheries (2008) 18:33–45

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Rev Fish Biol Fisheries (2008) 18:33–45 37

123

Amplified fragments were cut to detect polymor-

phisms using the following seven endonucleases:

TaqI, Sau3A1, RsaI, MspI, CfoI, Hinf I and BglII.

The first five enzymes recognize tetranucleotide

palindromic sequences, while the last two recognize

hexanucleotide sequences. Eight microliter of the

PCR product was incubated for 8 h with three units of

endonuclease in a final volume of 18 ml. The

fragments were separated in 1.5% agarose electro-

phoresis with TBE buffer and visualized using

ethidium bromide. In order to calculate molecular

size of fragments we used the comparison with

molecular size markers (500 bp). The RFLP pattern

produced by each endonuclease was identified with a

letter (Fig. 2), and each mtDNA haplotype was

defined by a code of seven letters (Table 2).

The ARLEQUIN program (Excoffier et al. 1992;

Schneider et al. 1997) was used to run the F tests,

haplotype frequency analyses, minimum spanning

tree. Genetic distances between haplotypes (Nei and

Li 1979) were determined using PAUP* 4.0 (Swof-

ford 2001). Presence/absence matrices of restriction

sites were processed with distance and parsimony

analysis (Dollo’s method) in PHYLIP 3.6 (Felsen-

stein 2005) and PAUP* 4.0.

Results

Haplotype distribution

A total of 23 mtDNA haplotypes were identified from

RFLP analyses of 15 wild trout populations (Table 3)

and four hatchery trout stocks (Table 4). RFLP

Fig. 2 Restriction fragment polymorphism patterns yielded by

the seven restriction enzymes for wild trout from northwestern

Mexico Ta

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38 Rev Fish Biol Fisheries (2008) 18:33–45

123

Ta

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AA

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FA

AA

A0.6

67

AA

BA

AA

A0.6

67

0.6

15

1.0

00

AB

BA

AA

A0.1

33

BA

BA

AA

A0.2

00

AC

AC

AA

A0.6

00

BC

AC

AA

A0.1

33

CC

AC

AA

A0.2

67

AA

AA

AA

B1.0

00

Rev Fish Biol Fisheries (2008) 18:33–45 39

123

patterns corresponding to each endonuclease are

depicted in Fig. 2. The size fragments under 160 bp

could not be reliably estimated.

The AAAAAAA haplotype is widespread among

the trout populations studied here, with exception of

six localities: Arroyo Verde, Mesa San Rafael, Casa

Quemada, La Quebrada, El Concheno, and Potrero de

Gil. The first three localities contain Mexican golden

trout. The three remaining sites contain unidentified

species of trout.

In this study, a total of 15 wild trout haplo-

types were not shared with hatchery trout samples.

One exclusive haplotype (AAAAEAA) was found in

O. mykiss nelsoni, from San Rafael and Santo

Domingo rivers in Sierra San Pedro Martir, Baja

California.

Three streams of the Rıo Yaqui were sampled and

in each one had 1 or 2 characteristic haplotypes

(BAAAAAA -La Presita-; AAAABAA —San Anto-

nio, Son-; AAEAACA and AAEACCA—Los Pesca-

dos-). Rıo Mayo in two localities had one pattern to

Potrero de Gil (AAAADAA) and two in El Concheno

(CCFABAA and ACFAAAA).

The O. chrysogaster in four localities, had one

haplotype shared with Arroyo Verde, La Onza and

Mesa San Rafael (AABAAAA), but this haplotype

was not found in Casa Quemada. This last site had

three exclusive patterns (ACACAAA, BCACAAA

and CCACAAA). Other patterns were also found in

Arroyo Verde (ABBAAAA and BABAAAA). The

southern trout had only one unique haplotype for the

La Quebrada site (Rıo Piaxtla). Other trout localities

showed hatcheries shared haplotypes.

Rainbow trout hatcheries in Mexico varied widely in

form and function. We visited Rancho San Antonio (Rıo

Yaqui), Ejido Vencedores (Rıo San Lorenzo), Los

Metates (Rıo Acaponeta) and Ejido La Victoria (Rıo

Presidio), as well as one inactive hatchery adjacent to

Arroyo La Presita (Rıo Yaqui). All had raceways

adjacent to streams with wild trout populations. Ejido

Vencedores and Ejido La Victoria have facilities for

breeding, incubation and rearing of young trout. In

contrast, rustic ponds built beside or in natural streams

were observed at Rancho Potrero de Gil (Rıo Mayo).

Haplotypes unique to hatchery specimens included:

CADAAAA and AAAAABA (Los Metates), ACA-

AAAA (Rancho Potrero de Gil and Ejido Vencedores),

and ACABAAA (Rancho San Antonio [Sonora],

Rancho Potrero de Gil and Ejido Vencedores). Three

other patterns (CAAAAAA, AACAAAA, and CCA-

BAAA) were shared between hatchery and wild trout in

this area. Haplotype AACAAAA was detected in

Rancho San Antonio [Sonora], Rancho Potrero de Gil

and Los Metates hatcheries with the highest frequency

in the latter population.

The CAAAAAA haplotype was observed fre-

quently in trout from the Rancho San Antonio

[Sonora], Rancho Potrero de Gil and Los Metates

hatcheries and it was also found in specimens from

streams adjacent to the hatcheries of Rancho San

Antonio (Sonora) and Coscomate (Rıo Baluarte), thus

indicating possible escape of cultured fish into to

natural streams. The CCABAAA haplotype was

detected in both wild and hatchery trout at the

localities of Rancho Potrero de Gil and Ejido

Vencederos (Rıo San Lorenzo).

Table 4 Haplotype frequencies for hatchery trout populations in northwestern Mexico

Hatchery

Basin Yaqui Mayo San Lorenzo Acaponeta

Haplotype San Antonio (H1) Potrero (H2) Vencedores (H3) Metates (H4)

N (14) (14) (13) (15)

AAAAAAA 0.357 0.5 0.692 0.600

CAAAAAA 0.214 0.143 0.200

ACABAAA 0.214 0.0714 0.154

AACAAAA 0.214 0.0714

CCABAAA 0.0714 0.077

ACAAAAA 0.143 0.077

CADAAAA 0.067

AAAAABA 0.133

40 Rev Fish Biol Fisheries (2008) 18:33–45

123

Haplotypes relationships

Using the minimum spanning network (MSN)

between all haplotypes (Fig. 3), the common

AAAAAA was related with others eleven by two or

three changes. From commonest, four exclusive

haplotypes groups in relation with geographical trout

distribution are indicated: (1) Nelson’s trout exclu-

sive haplotype, O. mykiss nelsoni, from the Rıo San

Rafael and Rıo Santo Domingo (AAAAEAA), (2)

Mayo and Yaqui trout, from the Arroyo Concheno

[Rıo Mayo] (ACFAAAA and CCFABAA), and some

trout from the arroyos Los Pescados and La Presita

(both in the Rıo Yaqui basin: BAAAAAA as well as

AAEAACA and AAEACCA), (3) Mexican golden

trout from the Rıo Fuerte and Rıo Culiacan basins

(AABAAAA, ABBAAAA and BABAAAA), and (4)

trout from the Rıo Piaxtla (AAAAAAB).

The hatcheries trout had more unique patterns

(AAAAABA, CAAAAAA, CADAAAA, AACAAAA,

ACAAAAA, ACABAAA and CCABAAA). In hatch-

ery trout, two haplotype groups were observed: the first

related to the unique patterns of the Rıo Mayo and

Rıo Sinaloa trout, and the second independent from

the patterns of the wild individuals as well as patterns

of the hatchery trout in Los Metates (Rıo Acaponeta).

When the shared or exclusives haplotypes of the

hatchery trout are excluded and reanalyzed the data

the grouping is maintained.

Neighbor joining trees, built from distances among

haplotypes and Dollo’s parsimony, produced sepa-

rated groups for all the wild trout populations from

northwestern Mexico. The dendrograms obtained by

different methods using all the identified haplotypes,

including those of the hatchery trout, showed differ-

ent grouping. The relationships among haplotypes

and populations were not consistently resolved in the

different analyses of branches displaying low consis-

tency, supporting politomies.

Statistical description

In this study, the diversity of restriction sites found in

wild trout populations ranked from 0 (monomorphic

in Arroyo Mesa San Rafael and Arroyo Piaxtla

populations), to 0.727 (in Arroyo Los Pescados), with

an average of 0.405. Estimate values of diversity

calculated according to Nei and Li (1979) among

populations are given in Table 5, along with observed

estimates of haplotypes nucleotide divergence. The

greatest estimated nucleotide divergence (0.049)

between wild trout occurred between BCACAAA

(O. chrysogaster) and CADAAAA (of Los Metates

hatchery), and between CCACAAA (O. chrysogaster

from Rıo Sinaloa) and BABAAAA (O. chrysogaster

from Rıo El Fuerte) (0.047). The minimum diver-

gence (d = 0.0033) was found between haplotypes of

O. mykiss from the Rıo Yaqui (AAAABAA) and the

Rıo Mayo (AAAADAA).

Discussion

The PCR–RFLP technique from animal mtDNA

could be applied to studies on genetic divergence of

AABAAAAACAAEAA

AAAAAAA

ACCAEAA

AAAAAAB

AADAAAA

AABAFCC

AAEAAAA

BAAAAAAAAAAAAC

AAAACAA

AAAABAA

AAAAFCA

AAAABBA

AAAABAB

AAACACA

AAACACB

AAACACC

)iuqaY(

)oyaM(

(Culiacán and Fuerte)

leafaRnaS(otnaSdna)ognimoD

(Piaxtla)

ABAAAAA

AAAADAC

AAABACA

AAABACC

AAAAACA

seirehctaH

(Sinaloa)

Fig. 3 Minimum spanning tree with the 23 mitochondrial

composite haplotypes from the Mexican wild and hatcheries

trout. Each line in the network represents a single mutational

change. One alternative connection is AAAAABA to AAEA-

ACA (three changes). Hatcheries haplotypes in dark boxes.

The wild trout haplotypes exclusives are indicated with river

names in parenthesis

Rev Fish Biol Fisheries (2008) 18:33–45 41

123

populations over large geographical areas, for study-

ing relationships among subspecies and higher

taxonomic units. The mtDNA genome have predom-

inantly maternal inheritance and relative high rate of

base-pair substitutions, especially in the D-Loop.

This non-coding region (D-Loop) is therefore a very

useful marker for the study of recently divergent

populations or species. A disadvantage of PCR–

RFLP of mtDNA is that the number of fragments may

be so large that fragments of similar size may not be

resolvable as separate fragments in an analysis

(Parker et al. 1998). In future studies, it will be

necessary to find the best enzyme.

To resolve the number of fragments to be used in

this work, we evaluated each fragment size by means

of contrasting with molecular size. We then com-

pared this with the constructed hypothetical patterns

of bands for each enzyme that were obtained from

the reports of Web data base of molecular genetic

data (Imsiridou et al. 2003) and GenBank (http://

www.ncbi.nlm.nih.gov), the mtDNA sequence of

O. mykiss (NC_001717) between cytochrome b (posi-

tion 15319–15343) and the D-loop position 1013–1033.

The presence of the common AAAAAAA haplo-

type in the wild and hatchery trout of northwestern

Mexico possibly represents a shared ancestral char-

acter or symplesiomorphy sequence series of O.

mykiss. This pattern would correspond to the theo-

retical haplotype for O. mykiss obtained from the

GenBank.

Studies focused on the genetic identification of

hatchery and wild haplotypes of Mexican trout in

northwestern Mexico, should help to improve the

management of this species complex to maintain the

genetic diversity of the native stocks; as other RFLP

studies have performed in Spain (Machordom et al.

2000), Italy (Caputo et al. 2004) or North America

(McCusker et al. 2000).

The exclusive haplotype (AAAAEAA) of O.

mykiss nelsoni from the Sierra San Pedro Martir

(SSPM), showed different proportions among locali-

ties and may be linked with the transplant history from

the original stock (Rancho San Antonio de Murillos)

to other localities into the same SSPM between 1929

and 1941 (Ruiz-Campos and Pister 1995).

In a previous study, Nielsen et al. (1997) identified

to the Mayo and Yaqui trout as one separate group

from Pacific trout on the basis of microsatellites and

mtDNA. In this work, RFLP haplotypes of these trout

forms were also differentiable from others. The three

Table 5 Values of genetic (Nei 1987) and nucleotide (Nei 1987; Tajima 1983) diversity in wild trout populations. For each locality

all haplotypes (wild, hatcheries and shared) are included in calculations

Locality Basin Genetic Diversity ± Nucleotide Diversity ±

San Rafael San Rafael 0.385 0.132 0.009 0.008

La Grulla Santo Domingo 0.303 0.148 0.007 0.007

San Antonio Santo Domingo 0.118 0.101 0.003 0.004

Arroyo Verde Fuerte 0.533 0.126 0.014 0.010

La Onza Fuerte 0.513 0.082 0.018 0.013

Mesa San Rafael Culiacan 0.000 0.000 0.000 0.000

Casa Quemada Sinaloa 0.591 0.106 0.015 0.011

La Presita Yaqui 0.385 0.132 0.009 0.008

San Antonio Yaqui 0.648 0.072 0.018 0.012

Los Pescados Yaqui 0.727 0.068 0.022 0.015

Potrero de Gil Mayo 0.363 0.130 0.042 0.025

El Concheno Mayo 0.667 0.314 0.031 0.028

Coscomate Baluarte 0.333 0.215 0.008 0.008

Los Metate Acaponeta 0.533 0.052 0.018 0.013

La Sidra San Lorenzo 0.385 0.132 0.031 0.020

La Quebrada Piaxtla 0.000 0.000 0.000 0.000

Mean 0.405 0.015

Standard Deviation 0.222 0.012

42 Rev Fish Biol Fisheries (2008) 18:33–45

123

individuals taken from Arroyo El Concheno (at Rıo

Mayo) had two exclusive haplotypes, however this

finding might not be representative of the total

population due to the small sample size.

Two other localities of the Rıo Yaqui basin; such

as La Presita and Los Pescados arroyos, had exclusive

(per locality) and common haplotypes. However, it is

important to note the presence of a hatchery instal-

lation next to La Presita site, which is in temporary

use and might therefore represent a potential risk of

escapes for hatchery trout. The haplotype distribution

of the two sites already referred to, may show

different isolation histories and geographic barriers.

In the trout samples from the Rıo Yaqui (Arroyo San

Antonio) and Rıo Mayo (Potrero de Gil), both

collected in sites adjacent to trout hatcheries, the

examined individuals had shared haplotypes with

those of the hatchery. However we cannot assign

them as wild or hatcheries trout, because the PCR–

RFLP of mtDNA with maternal transmission (Parker

et al. 1998) is limited to realize this exclusion.

All the haplotypes found in Mexican golden trout

(O. chrysogaster), in four localities, were exclusive

and only the La Onza site located the commonest

AAAAAAA haplotype. One haplotype (AABAAAA)

was found in three localities and will be used as a

molecular marker for this species. Finally, the four

southern localities are recognized by the frequent

hatchery shared haplotypes, although only in La

Quebrada had one exclusive haplotype.

Eight haplotypes found in hatchery trout from

Mexico provided direct evidence for the current diver-

sity of Mexican cultured stocks and could be the result of

more than one source population for introduced hatch-

ery stocks. Hybridization between hatchery and wild

trout has been reported in rivers of California (Behnke

1991; Nielsen et al. 1999; Young et al. 2001; Ostberg

and Rodrıguez 2002) and Europe (Machordom et al.

1999; Hansen et al. 2000; Bernatchez 2001). Such

findings led us to expect hybridization in Mexican trout.

However, complementary genetic studies using nuclear

markers, as VNTRs recommended by Parker et al.

(1998), are needed in order to evaluate additional details

of hybridization of Mexican native and hatchery.

The localities adjacent to hatcheries or with shared

hatchery haplotypes, will have hybridization risk

and the introduced exotic genotypes may reduce genetic

differentiation among populations, if the same exotic

types are introduced into several populations.

To reduce the risk of hybridization and loss of

genetic differentiation, it is important to implement

and enforce well designed management plans that

will assure conservation of the diversity of Mexican

native trout, as well as the protection of their habitats.

The following streams, referred from north to south,

will be key to wild trout conservation because of the

absence of hatcheries near the streams with wild

trout: (a) San Rafael and Rıo Santo Domingo for

Nelson’s trout; (b) Arroyo Los Pescados and Arroyo

El Concheno for Yaqui and Mayo trout, respectively;

(c) Arroyo Verde, Arroyo La Onza, Arroyo Mesa San

Rafael and Arroyo Casa Quemada for Golden

Mexican trout; and finally (d) Arroyo La Quebrada

for Rıo Piaxtla trout.

Two groups are currently protected in Mexico with

legal statements as NOM-059: the Nelson’s trout and

the Golden Mexican trout, however groups from the

following localities: Los Pescados, El Concheno and

La Quebrada, do not have any special protection and

would be important in Mexican trout conservation

programs. To protect these areas would be complex

because there is forest exploitation and are in

communal property. Another way would be to protect

the species or subspecies as a species in danger of

extinction (SEMARNAT 2002). In this way, the first

step would be describe this trout with the taxonom-

ical rules and establish its nomenclatural status.

Conclusions

Analyses of restriction of haplotypes for amplifica-

tions of the fragment between cytochrome b and D-

Loop digested with seven endonucleases, identified a

number of unique haplotypes for O. mykiss nelsoni,

O. chrysogaster and the undescribed trout of the Rıo

Yaqui, Rıo Mayo and Rıo Piaxtla basins.

Fifteen wild trout haplotypes identified here were

classified in four groups:

(1) haplotype AAAAEAA restricted to O. mykiss

nelsoni of the Sierra San Pedro Martir;

(2) haplotypes in populations of the Rıo Yaqui

(BAAAAAA, AAAABAA, AAEAACA,

AAEACCA) and Rıo Mayo (AAAADAA,

CCFABAA, ACFAAAA) basins;

(3) haplotypes exclusive to O. chrysogaster, with

two sub-groups (Rıo Sinaloa: ACACAAA,

BCACAAA, CCACAAA; and Rıo Culiacan

Rev Fish Biol Fisheries (2008) 18:33–45 43

123

and Rıo Fuerte: AABAAAA, ABBAAAA,

BABAAAA);

(4) haplotype AAAAAAB exclusive to the Rıo

Piaxtla

(5) haplotype (AAAAAAA) had a wide distribution

in trout populations (both wild and hatcheries).

The distribution of haplotypes in wild trout

populations from northwestern Mexico reflected the

management history for the cultured rainbow trout,

which facilitates the possibility of escape of cultured

rainbow trout to streams occupied by native and/or

the mixing of native trout with introduced trout from

outside of Mexico in culture systems.

It is important to implement and enforce well

designed management plans that will ensure conserva-

tion of the diversity of Mexican native trout, as well as

the protection of their habitats. This will entail instal-

lation of devices in culture facilities to prevent escapes

and implementation of various other security measures

in the trout culture industry to avoid or reduce the

escapes of individuals to the adjacent streams.

Acknowledgements Numerous people participated in the

trout sampling and are greatly acknowledged. Our special

thanks to the field guides B. Felix, I. Garcıa and F. Garcıa

(Mesa Tres Rıos, Sonora), S. Camunez, A. Paredes and J.

Navarro (Basaseachic, Chihuahua), L. Saavedra and J.

Escarcega (Guadalupe and Calvo, Chihuahua), L. A. Rıos (El

Salto, Durango), I. Rodrıguez and A. Aguilar (San Miguel de

Cruces, Durango). D. A. Hendrickson and B. Jensen provided

information on collecting sites in SMO. N. Villarreal, J.

Echanove, C. Brum, U. Pacheco, A. Jullian, F. Leon and J.

Zamudio supported the trout sampling in the SSPM. This work

was supported by the Consejo Nacional de Ciencia y

Tecnologıa (agreement CONACYT 33528-V) and the

Universidad Autonoma de Baja California. The trout

collecting permit was authorized by the Secretarıa de

Agricultura, Ganaderıa, Desarrollo Rural, Pesca y

Alimentacion (permit number 060201–613-03). Jennifer L.

Nielsen and three anonymous reviewers made useful comments

and recommendations that significantly improved the content

of the manuscript.

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