Journal of Heredity 2014:105(1):91–100
doi:10.1093/jhered/est067
Advance Access publication October 11, 2013
© The American Genetic Association 2013. All rights reserved.
For permissions, please e-mail: [email protected]
Strong Population Structure and
Shallow Mitochondrial Phylogeny in the
Banded Guitarfish, Zapteryx exasperata
(Jordan y Gilbert, 1880), from the
Northern Mexican Pacific
Ana Castillo-Páez, Oscar Sosa-Nishizaki, Jonathan Sandoval-Castillo,
Felipe Galván-Magaña, María-del-Pilar Blanco-Parra, and Axayácatl Rocha-Olivares
From the Department of Biological Oceanography, Molecular Ecology Laboratory, CICESE, Carretera Ensenada-Tijuana
No. 3918, Ensenada, Baja California 22860, México (Castillo-Páez and Rocha-Olivares); the Department of Biological
Oceanography, Fisheries Ecology Laboratory, CICESE, Carretera Ensenada-Tijuana No. 3918, Ensenada, Baja California 22860,
México (Sosa-Nishizaki); the Molecular Ecology Laboratory, School of Biological Sciences, Flinders University, Adelaide, SA
5049, Australia (Sandoval-Castillo); the Departamento de Pesquerías y Biología Marina, Laboratorio de Ecología de Peces,
Centro Interdisciplinario de Ciencias Marinas, IPN, Av. Instituto Politécnico Nacional s/n, Col. Playa Palo de Santa Rita, La Paz,
Baja California Sur 23096, México (Galván-Magaña); and the División de Ciencias e Ingeniería, Universidad de Quintana Roo,
Boulevard Bahía s/n. Esquina Ignacio Comonfort, Colonia del Bosque, Chetumal, Quintana Roo, México (Blanco-Parra).
Address correspondence to Axayácatl Rocha-Olivares at the address above, or e-mail: [email protected].
Data deposited at Dryad: http://dx.doi.org/10.5061/dryad.k60kb
Abstract
The northern Mexican Pacific (NMP), the Gulf of California (GC), and Baja California have been recognized as an ecological and evolutionarily dynamic region having experienced significant tectonic and climatic changes leading to the diversification of terrestrial and marine biotas. Zapteryx exasperata is a predominant ray caught in the artisanal fisheries of the NMP.
Morphometric and reproductive differences between rays from the GC and the Pacific coast of Baja California (PCBC) regions
suggest the presence of distinct populations. We investigate whether this distinction correlates with differences in genetic
diversity and differentiation using sequences of the mitochondrial nicotinamide adenine dinucleotide dehydrogenase subunit 2
(ND2) gene and the noncoding control region (CR) in 63 specimens. Contrary to our expectations, ND2 bore significantly more
diversity (h = 0.76) than CR (h = 0.39). Geographic patterns of diversity of CR were opposite to those of ND2, with GC being
significantly less (ND2) and more (CR) diverse than PCBC. The diversity of concatenated haplotypes was high (h = 0.84). Low
nucleotide diversity suggests the recent coancestry of haplotypes. Marked genetic structure (Φst = 0.23, P < 0.0001) revealed
the existence of reproductive isolation and limited matrilineal gene flow between GC and PCBC, which correlates with their
phenotypic distinction. These results suggest the influence of factors such as female reproductive philopatry, and ecological or
historical vicariant barriers to gene flow. Our results point to the existence of a distinct management unit of banded guitarfish
in each region, and add to the increasing evidence attesting to the diversifying nature of this evolutionarily dynamic region.
Key words: Baja California, elasmobranch, genetic diversity, Gulf of California, mitochondrial DNA
The northern Mexican Pacific (NMP) is an ecologically and
evolutionarily dynamic region. It features a rich assemblage
of biological diversity that has evolved under a scenario
of active tectonic activity and climatic oscillations, which
have shifted the transitional frontier between temperate
and tropical communities during the last few million years
(reviewed by Jacobs et al. 2004). Tectonic activity is responsible for the origination of the peninsula of Baja California
and the Gulf of California (GC) around 6 million years ago
(mya). The complex physiographic changes leading to its
91
Journal of Heredity 
actual configuration around 3.5 mya produced vicariant isolation of marine faunas by the peninsula, as well as episodes
of contact and dispersal between the Pacific and gulf by
means of seaways running across the developing peninsula
(Upton and Murphy 1997; Riddle et al. 2000). This scenario
of shifting continental plates and climatic oscillations during
the Miocene and Pleistocene set the stage for the evolutionary diversification of the marine and terrestrial biotas of the
region (Riddle et al. 2000; Bernardi et al. 2003; Jacobs et al.
2004; Riginos 2005; Segura et al. 2006).
In spite of their ecological and evolutionary significance,
the genetics of cartilaginous fishes remain understudied
in the vast majority of the world’s oceans. Among elasmobranchs, batoids (rays) have received even less attention than
their shark relatives. In these organisms, the existence of
genetic heterogeneity has been attributed to several factors
such as the reproductive philopatry promoting isolation of
geographical reproductive groups, as in Raja clavata (Chevolot
et al. 2006a) and Pristis microdon (Phillips et al. 2011); oceanographic conditions promoting ecological isolation and cryptic
speciation, as in Dipturus batis in which temperature gradients and thermal physiological limits affect the distribution
of geographic clades (Griffiths et al. 2010); and the presence
of geographic barriers (i.e., vicariance), such as in populations inhabiting both coasts of the Baja California peninsula.
Several genetic surveys have revealed a phylogeographic break
of mitochondrial lineages and a genetic distinction between
batoids residing in the GC relative to those in the Pacific coast
of Baja California (PCBC). These include the shovelnose guitarfish Rhinobatos productus (Sandoval-Castillo et al. 2004), the
golden cownose ray Rhinoptera steindachneri (Sandoval-Castillo
and Rocha-Olivares 2011), and the California butterfly ray
Gymnura marmorata (Smith et al. 2009), which raises the question of how widespread among batoids is the diversifying
influence of the region on their population genetic structure.
The study of shared patterns of genetic differentiation and
genealogic concordance of phylogroups is of fundamental
importance to unravel the ecological and evolutionary forces
that have shaped the genetic makeup of sympatric species and
to understand the nature of genetic discontinuities within an
otherwise continuous distribution (Avise 1992, 2000).
The focus of this study is the banded guitarfish, Zapteryx
exasperata, a demersal ray continuously distributed from
southern California to Peru, including the GC (McEachran
and Notarbartolo di Sciara 1995; Ebert 2003). Banded guitarfish is a predominant ray in the elasmobranch catches of
artisanal fisheries carried out in the NMP (Blanco-Parra et al.
2009a; Cartamil et al. 2011), but little is known about its biology. The available information points to the phenotypic distinction of individuals from the GC and the PCBC. These
include morphometric and reproductive differences. Rays
landed in Bahía Vizcaino (PCBC) are on average significantly
larger than those from Sonora (GC) (Blanco-Parra et al.
2009a; Cartamil et al. 2011). In addition, female gestation and
vitellogenesis in GC rays are concurrent; following copulation, embryos undergo a developmental arrest (diapause) and
are born during the subsequent summer (Blanco-Parra et al.
2009b). In contrast, gestation and oocyte development in rays
92
from the PCBC (Bahía Almejas) are not concurrent; oocyte
development peaks in March, prior to copulation, ­gestation,
and the ensuing parturition, which takes place during the
summer of the same reproductive season (VillavicencioGarayzar 1995). Hence, we address the question of whether
the observed phenotypic differences above relate to genetically distinct allopatric populations or are manifestations
of phenotypic plasticity in the face of contrasting ecological regimes between the PCBC and the GC. We employed
2 mitochondrial (mtDNA) gene regions, a structural gene
(nicotinamide adenine dinucleotide dehydrogenase subunit 2
or ND2) and the noncoding control region (CR) to assess
levels of genetic diversity and heterogeneity. These molecular data revealed significant levels of differentiation between
gulf and Pacific rays, which may result from behavioral traits
such as fidelity to breeding sites and the existence of physical and oceanographic/ecological barriers to dispersal. These
findings add to the increasing evidence attesting to the diversifying nature of this evolutionarily dynamic region.
Materials and Methods
Sampling
Muscle tissue samples of Z. exasperata were collected from
11 artisanal fishing camps, 5 along GC (Estero del Soldado
27°95′N, 110°96′W; Bahía Kino 28°82′N, 111°94′W; San
Felipe 31°01′N, 114°83′W; Bahía de los Angeles 8°95′N,
113°56′W; Bahía de la Paz 24°15′N, 110°31′W) and 6 from the
PCBC (Puerto San Carlos 29°63′N, 115°48′W; Punta Canoas
29°42′N, 115°12′W; Punta Santa María 28°94′N, 114°51′W;
Laguna Manuela 28°24′N, 114°08′W; Guerrero Negro
28°02′N, 114°12′W, and San Ignacio 26°38′N, 113°18′W)
(Figure 1). Due to the scatter of the geographic of sample
sites and the small sample sizes per camp, we analyzed the
data in 5 samples (3 in the region of PCBC and 2 in GC) that
grouped fishing camps by proximity, with the exception of
the Western Gulf sample, which groups all individuals collected in the western coast of the GC (Figure 1, Table 1).
Tissues were preserved in nondenatured ethanol (95%) until
processed in the laboratory.
DNA Extraction, Amplification, and Sequencing
Genomic DNA was extracted using approximately 70–100 mg
of finely chopped muscle tissue, using proteinase K digestion followed by a salting-out protocol with lithium chloride
(Gemmell and Akiyama 1996). Two mitochondrial (mtDNA)
genes were amplified, ND2 using primers ND2Met47 and
ND2Trp18 (Sandoval-Castillo and Rocha-Olivares 2011) and
CR using primers ZECR-H (GTG TCT CCG TGG TCC
AAC) and ZECR-L (GGT CAG ATT AAC ATA TAA TGT
ATA TAG CC) designed for this study. Each 25 µL containing 0.18 mM of dNTPs, 1× polymerase chain reaction (PCR)
buffer (200 mM Tris HCl pH 8.3, 100 mM (NH)4SO4, 100 mM
KCl, 20 mM MgSO4, 1% Triton x-100), 0.28 μM of each
primer, 0.75 U Taq DNA-pol (BioLabs, Ipswich, MA), 0.4 mg/
mL bovine serum albumin (BioLabs), and 20 ng of template
Castillo-Páez et al. • Strong Population Structure in Banded Guitarfish
Figure 1. Sampling localities of Zapteryx exasperata corresponding to artisanal fishing camps. Pie charts represent frequency of
concatenated haplotypes (identified by different fillings and numbers). n, sample size; PSC, Puerto San Carlos; PC, Punta Canoas;
PSM, Punta Santa María; LM, Laguna Manuela; GN, Guerrero Negro; SI, San Ignacio; SF, San Felipe; AB, Bahía de los Ángeles;
PB, Bahía de La Paz; KB, Bahía Kino; SE, Estero del Soldado.
Table 1 Levels of mitochondrial diversity in mitochondrial ND2, CR, and CAT of Zapteryx exasperata from the GC and the PCBC
Samples (year of collection)
Gene
ND2
CR
CAT
n
A
h
π
A
h
π
A
h
π
Regions
PC (2011)
BV (2011)
SI (2002)
SO (2004)
WG (2002)
PCBC
GC
Total
10
4
0.778
0.0016
1
0.000
0.0000
4
0.778
0.0010
16
4
0.650
0.0015
1
0.000
0.0000
4
0.650
0.0009
11
6
0.800
0.0020
2
0.327
0.0005
6
0.800
0.0014
22
3
0.255
0.0004
2
0.504
0.0009
5
0.641
0.0006
4
2
0.500
0.0010
2
0.500
0.0007
3
0.833
0.0009
37
8
0.721
0.0016
2
0.105
0.0002
8
0.721
0.0011
26
4
0.286
0.0005
3
0.557
0.0010
7
0.692
0.0007
63
10
0.760
0.0013
3
0.387
0.0007
13
0.840
0.0011
n, sample size; A, number of haplotypes; h, haplotype diversity; π, nucleotide diversity; PC, Punta Canoas; BV, Bahía Vizcaíno; SI, San Ignacio; SO, Sonora;
WG, Western Gulf.
93
Journal of Heredity 
DNA. All reactions included an experimental negative control.
Thermal cycling included an initial denaturation of 5 min at
95 °C, followed by 35 cycles for 15 s at 95 °C, 30 s at 60 °C
(ND2) or 62 °C (CR) of primer annealing and 2 min at 72 °C;
and final elongation for 10 min at 72 °C. PCR products were
purified using the QIAquick PCR Purification Kit (QIAGEN,
Valencia, CA) according to the manufacturer’s instructions and
then sequenced using PCR primers and Big Dye Terminator
v.3.1 chemistry in an ABI 3730xl (Applied Biosystems-Life
Technologies, Grand Island, NY).
Sequence Analyses
After trimming noisy ends of sequences, base calling was
confirmed by visually inspecting chromatograms with
CodonCode Aligner v 3.7.1, (Codon Code Corporation,
Dedham, MA). Genes were analyzed separately and concatenated. All sequences were aligned using the algorithm
CLUSTAL W as implemented in MEGA v 5.05 (Tamura
et al. 2011). Haplotypes were identified using DNAsp v 5
(Librado and Rozas 2009). Haplotype (h) and nucleotide (π) diversities were estimated using Arlequin v 3.5.1.3
(Excoffier and Lischer 2010). Hutcheson’s t-test was used to
compare diversity indexes, and Spearman’s coefficient was
used for correlations between diversity indices (Zar 1984).
Population structure was assessed by analysis of molecular
variance (AMOVA) using Arlequin (Excoffier and Lischer
2010), in which a distance of matrix model-based genetic
distances was used as Euclidean distances. Distances were
computed using PAUP* v.4 (Swofford 2002) using the bestfit model of molecular evolution (HKY+I for ND2, HKY
for CR, and TrN+I for concatenated haplotypes, selected
following the corrected Akaike Information Criterion) as
estimated by jModelTest v 2.0.2 (Posada 2008). Pairwise
molecular fixation indexes (Φst) were estimated to assess differences between population pairs using Arlequin v 3.5.1.3
(Excoffier and Lischer 2010). The sequential Bonferroni correction was used to adjust significance for multiple tests (Rice
1989). A first-order approximation of gene flow (Nm) levels
was obtained from paired Φst values assuming Wright’s island
model (Wright 1951). We also attempted to estimate mutation-scaled levels of effective population size (θ = 2 Neµ)
and gene flow (M = m/µ) using a model-based approach
involving maximum likelihood Markov Chain Monte Carlo
(MCMC), as implemented in MIGRATE v.2.1.3 (Beerli
2004). We used 10 short-chain searches (5 000 genealogies),
3 long-chain searches (50 000 genealogies) and a burn-in of
10 000 trees to ensure independence from initial conditions.
Phylogeographic Analysis
Phylogenetic relationships among mtDNA haplotypes were
estimated for separate and concatenated haplotypes with an
unrooted statistical parsimony network using the software
package TCS v 1.2.1 (Clement et al. 2000), run with the
default probability connection limit (95%).
In compliance with data archiving guidelines (Baker
2013), we have deposited the primary data sets underlying
these analyses with Dryad.
94
Results
Patterns of Genetic Diversity
Mitochondrial DNA sequences revealed very modest levels
of molecular diversity. The 984 base pair (bp)-long ND2
alignment of 63 individuals contained only 8 polymorphic
sites. All mutations were transitions and resolved 10 haplotypes (4 shared and 6 private; see Electronic Supplementary
Material Table S1, and GeneBank accession numbers:
KC610072–KC610081). Haplotype ZEND2-1 was found
throughout the study area; however, it was predominant
in the GC (found in 85% of individuals). ZEND2-3 was
the second most frequent haplotype and was found exclusively in the PCBC localities (see Electronic Supplementary
Material Figure S1). The highest number of private haplotypes was found in San Ignacio (ZEND2-6, ZEND2-7, and
ZEND2-8). Levels of ND2 haplotype diversity were highly
variable (Table 1), ranging from h = 0.80 in San Ignacio to
h = 0.26 in Sonora. Haplotype diversity was significantly
larger in the PCBC (h = 0.72) than in the GC (h = 0.29)
(t = 3.37, P = 0.002). Nucleotide diversity was low in all
localities, correlated with haplotype diversities (Spearman’s
correlation rs = 0.99), and was not significantly different
between the 2 regions (t = 0.90, P = 0.37), reflecting a low
degree of genetic divergence among mitochondrial ND2
gene sequences.
Extremely low levels of genetic diversity were found in
the CR. The 604 bp-long alignment of CR sequences contained only 2 polymorphic sites (also transitions), which produced 3 haplotypes, 2 common and 1 private to the GC (see
Electronic Supplementary Material Table S1 and GeneBank
accession numbers: KC610069–KC610071). Localities were
fixed for a mitochondrial haplotype, and the most diverse
samples were San Ignacio (h = 0.33 and π = 0.0005) on the
PCBC; in the GC, both localities had the same diversity
(Table 1). In contrast to ND2, rays from the GC (h = 0.56)
were significantly more diverse than those from the PCBC
(h = 0.11; t = 5.69, P < 0.001; see Electronic Supplementary
Material Table S1). With only 3 weakly divergent haplotypes,
nucleotide diversity was an order of magnitude smaller than
the one found in ND2 and not significantly different between
the 2 regions (t = 0.83, P = 0.41).
Concatenated haplotypes produced a 1588 bp alignment,
containing 10 variable sites, which produced 13 haplotypes
(4 shared and 9 private; Figure 1, Table 2). For the PCBC,
highest number of private haplotypes and the most diverse
samples were San Ignacio (h = 0.800 and π = 0.0014); haplotypes ZAEX_6 and ZAEX_8 were exclusive in this region
(Figure 1). For the GC, highest number of private haplotypes were Sonora with 4, but west cost of the GC (Western
Gulf) was the most diverse samples (h = 0.83 and π = 0.0009;
Table 1). Haplotype (t = 0.32, P = 0.75) and nucleotide
(t = 0.05, P = 0.96) diversity was not significantly different
between the 2 regions. Diversity levels from samples on the
west cost of the GC (Western Gulf sample) are grouped for
informative purposes only and should interpreted with caution given the small sample size (n = 4) and the fact that they
are not from a single locality.
Castillo-Páez et al. • Strong Population Structure in Banded Guitarfish
equivalent to approximately 1 or 2 female migrants every 3
generations (Table 4). Despite numerous attempts to obtain
acceptable results from the program MIGRATE, MCMC
runs yielded widely variable and inconsistent results pointing to a lack of convergence of the sampler. Presumably, the
mtDNA data lacked sufficient variation for the estimation of
all model parameters.
Population Structure and Gene Flow
Both mitochondrial genes exhibited a significant genetic
structure overall (Φs t -ND 2 = 0 . 16, P < 0.0001; Φs t -C R = 0 . 4 2,
P < 0.0001; Φs t -C A T = 0 . 2 3, P < 0.0001), and a high percentage of the genetic variance was partitioned between
the geographic regions (Table 3). Highly significant pairwise Φst values in both mitochondrial genes revealed that
genetic differences resided primarily between Sonora (GC)
and PCBC localities. No differentiation was found among
PCBC localities owing to high levels of gene flow. Average
gene flow between the gulf and the Pacific was limited (NmND2 = 0.65, Nm-CR = 0.33, and Nm-CAT = 0.47) and was
Phylogeography
The frequencies of mtDNA haplotypes are clearly geographically heterogeneous with concatenated genes; the same
pattern was found in each gene separately (see Electronic
Table 2 Polymorphic nucleotides of concatenated haplotypes of Zapteryx exasperata from the GC and the PCBC
Variable sites
Haplotypes
3
92
251
431
653
820
836
859
1191
1247
ZAEX_1
ZAEX_2
ZAEX_3
ZAEX_4
ZAEX_5
ZAEX_6
ZAEX_7
ZAEX_8
ZAEX_9
ZAEX_10
ZAEX_11
ZAEX_12
ZAEX_13
T
.
.
C
.
C
C
.
.
C
.
.
C
T
.
.
.
.
.
.
.
.
C
.
.
C
C
.
.
.
.
.
T
.
.
.
T
.
.
C
.
.
.
.
T
T
.
.
.
.
.
.
C
T
.
.
.
.
.
.
.
.
.
.
.
C
T
.
.
T
.
.
.
T
.
.
.
.
C
.
.
.
.
.
.
.
T
.
.
.
.
G
.
.
.
.
.
.
A
.
.
.
.
.
T
.
.
.
.
.
.
C
.
.
.
C
.
T
.
C
.
.
.
.
.
.
C
.
.
.
Table 3 AMOVA for ND2, CR, and CAT genes of mtDNA of Zapteryx exasperata
Gene
Source of variation
df
Sums of squares
Variance components
Percent variation
ND2
Among samples
Within samples
Total
Among samples
Within samples
Total
Among samples
Within samples
Total
3
55
58
3
55
58
3
55
58
0.007
0.032
0.039
0.007
0.012
0.019
0.007
0.025
0.032
0.0001
0.0006
0.0007
0.0001
0.0002
0.0004
0.0001
0.0005
0.0006
16.59
83.41
CR
CAT
42.01
57.99
23.27
76.73
df, degrees of freedom.
Table 4 Pairwise values of the fixation index (Φst) (below the diagonal) and gene flow (Nm) between pairs of samples (above the
diagonal) based on ND2, CR, and CAT DNA sequences
SO
PC
Samples
ND2
CR
CAT
SO
PC
BV
SI
0.337
0.333
0.209
0.427
0.481
0.391
0.291
0.386
0.375
ND2
BV
CR
CAT
0.492
0.336
0.416
−0.026
0.0312
0.000
0.088
−0.007
0.037
SI
ND2
CR
CAT
ND2
CR
CAT
0.501
∞
0.269
∞
0.397
∞
−0.014
0.150
−0.027
0.945
7.607
∞
0.390
2.594
1.422
0.608
6.399
∞
Significant P values (P < 0.05) after sequential Bonferroni correction adjustment are shown in bold. SO, Sonora; PC, Punta Canoas; BV, Bahía Vizcaíno; SI,
San Ignacio.
95
Journal of Heredity 
Supplementary Material Figure S2). This produced a strong
genetic structure between PCBC and GC; however, the phylogenetic relationships among haplotypes were extremely
close and did not reveal the existence of reciprocally monophyletic allopatric lineages. On the other hand, the geographic distribution of the most abundant haplotypes, being
almost private to a region, may be construed as an incipient
phylogeographic pattern. Concatenated haplotypes differed
mostly by 2 or 3 mutations (Figure 2). Haplotypes ZAEX6, ZAEX-9, and ZAEX-10 found in PCBC are connected
by a mutational step; similarly, the positions in the network
of ZAEX-8 and ZAEX-11 suggest their recent evolutionary
divergence.
Discussion
Genetic Diversity
We provide the first genetic diversity assessment of the
banded guitarfish Z. exasperata in the NMP. Previously, the
CR has often been found to be the most polymorphic mitochondrial region across a number of divergent vertebrate
taxa (Lee et al. 1995; Zhang et al. 1995; Lopez et al. 1997;
McMillan and Palumbi 1997; Rocha-Olivares et al. 1999);
hence, it has been the marker of choice for innumerable
studies, including several elasmobranch population genetics R. productus (Sandoval-Castillo et al. 2004), hammerhead
sharks, Sphyrna spp. (Quattro et al. 2005), Carcharhinus limbatus (Keeney and Heist 2006), Rhincodon typus (Castro et al.
2007), Carcharias taurus (Ahonen et al. 2009), Galeorhinus galeus
(Chabot and Allen 2009), D. batis (Griffiths et al. 2010), and
Triaenodon obesus (Whitney et al. 2012). Contrary to our expectations, we found more variation in the structural gene ND2.
To our knowledge, this is the first time this odd pattern of
variation has been found in a batoid. Previously, it has only
been reported in sharks: in the zebra shark Stegostoma fasciatum in the Indo-West Pacific, in which the CR was also the
least variable among 3 other structural genes (COI, ATPase,
and ND4; Dudgeon et al. 2009); in the gummy shark Mustelus
antarcticus in the central Indo-Pacific and Australasia, in which
ND4 showed greater variability than the monomorphic CR in
10 individuals collected across its distribution range (Boomer
et al. 2012); and it was also found in Orectolobus sharks (ND4
greater variability than CR; S. Corrigan et al. unpublished
data), all of which are epibenthic elasmobranchs. This odd
pattern of molecular evolution warrants further study, particularly to investigate its underlying causes, which could
include increased evolutionary constraints on this gene segment and historical selective sweeps, the existence of undetected paralogous gene copies (NUMTs, Richly and Leister
2004), or other structural changes in the mitochondrial
genome. In addition, it remains to be established how common it is among cartilaginous fishes. On the other hand, evidence of the utility of the ND2 gene to resolve intraspecific
and shallow interspecific relationships in elasmobranchs has
been increasing (Naylor et al. 1997, 2012).
Low to moderate levels of genetic polymorphisms in the
elasmobranch CR have been linked to their relatively slow
rate of molecular evolution compared with other vertebrates
(Martin et al. 1992). Values found in the PCBC are among the
lowest reported for batoid species (Table 5) and comparable
with those found in the CR of Raja miraletus and Raja asterias in
the Mediterranean (Valsecchi et al. 2005). On the other hand,
higher levels found in ND2 are similar to those reported in
R. clavata (the Eastern Atlantic; h = 0.54; Pasolini et al. 2011),
Pristis clavata and Pristis zijsron (Northern Australia; Phillips et al.
Figure 2. Statistical parsimony network of mitochondrial concatenated haplotypes of Zapteryx exasperata. Circle size is
proportional to abundance according to scale shown. Numbers represent haplotypes, and their frequency in both geographic
regions (PCBC and GC) is symbolized as a pie chart. Lines connecting haplotypes represent single mutational steps.
96
Castillo-Páez et al. • Strong Population Structure in Banded Guitarfish
Table 5 Genetic diversity of mtDNA in batoid species
Species
Gen
Geographic region
N
A
h
π
Reference
Aetobatus narinari
Cyt-b
48
12
41
3
25
30
11
16
2
30
240
67
8
152
10
6
73
149
49
18
26
12
38
22
27
52
42
18
30
25
32
32
38
38
4
5
3
2
4
6
7
2
2
10
27
14
6
8
3
7
15
18
9
2
6
2
6
6
5
13
12
7
9
8
9
8
2
2
0.1968
0.7273
0.2976
0.6667
0.6200
0.4943
0.8909
0.1250
1.0000
0.834
0.810
0.688
0.93
0.73
0.58
1.00
0.489
0.650
0.555
0.29
0.61
0.17
0.578
0.411
0.450
0.521
0.460
0.843
0.782
0.490
0.749
0.794
0.152
0.000
0.0004
0.0030
0.0007
0.0055
0.0015
0.0025
0.0037
0.0007
0.0029
0.0070
0.0053
0.0040
0.0019
0.0006
0.0003
0.0011
0.0040
0.0044
0.0036
0.0092
0.0072
0.0031
0.0027
0.0021
0.0014
0.0018
0.0020
0.0013
0.0034
0.0013
0.0106
0.0131
0.503
0.000
(Schluessel et al. 2010)
Amblyraja radiata
Cyt-b
Dasyatis brevicaudata
CR
Pristis clavata
Pristis microdon
Pristis zijsron
Raja asterias
Raja clavata
Raja miraletus
Raja clavata
CR
Australia
Southeast Asia
East China Sea
Central Pacific
Atlantic
Australia
Southeast Asia
East China Sea
Atlantic
Newfoundland
Iceland
Eastern Atlantic
Eastern Australia
New Zealand
South Africa
Western Australia
Northern Australia
CR
Mediterranean
CR
Raja straeleni
CR
Rhinobatos productus
CR-RFLP
Rhinoptera steindachneri
ND2
North Sea (Europe)
Irish Sea (Europe)
Algerian Coasts (Europe)
Tyrrhenian Sea (Europe)
Adriatic sea (Europe)
Eastern Mediterranean
Cape town (Africa)
South coast (Africa)
Gulf of California
Pacific coast
Gulf of California
Pacific coast
ND4
2011). However, high levels of molecular diversity were found
in the sympatric and more closely related shovelnose guitarfish
R. products (Sandoval-Castillo et al. 2004), probably due to the
existence of cryptic species and larger population sizes. Low
levels of ND2 haplotype diversity have been found in sympatric R. steindachneri from the GC (Table 5; Sandoval-Castillo
and Rocha-Olivares 2011); however, these results involved
the identification of haplotypes by means of lower resolution
restriction fragment length polymorphisms (RFLPs). On the
other hand, diversity levels found in the PCBC are comparable
to those of other structural genes such as ND4 for Aetobatus
narinari in Southeast Asia (h = 0.89; Schluessel et al. 2010).
Notably, both mtDNA genes not only differed in levels of molecular diversity but also in their patterns of geographic variation; therefore, it is premature to conclude
which geographic region is genetically less diverse without
additional data. Rays from the GC had higher CR but lower
ND2 diversity levels than those from the Pacific, even though
a comparable number of individuals were analyzed. The
smaller ND2 diversity from the GC fishes may relate to a
narrower geographic origin of the samples (85% of the GC
are from Sonora), to the presence of higher levels of genetic
drift and hence smaller effective population size in the gulf
(Chevolot et al. 2007)
(Le Port and Lavery 2012)
(Phillips et al. 2011)
(Valsecchi et al. 2005)
(Pasolini et al. 2011)
(Pasolini et al. 2011)
(Sandoval-Castillo et al. 2004)
(Sandoval-Castillo and
Rocha-Olivares 2011)
(Le Port and Lavery 2012), or to the presence of stronger
selective pressures reflected in the mtDNA. These factors
could maintain differences in the levels of genetic diversity
in the presence of limited gene flow. The potentially low
genetic diversity of GC guitarfishes needs to be corroborated
with more intense genetic and geographic sampling, as it may
impinge on the vulnerability of the species to face and survive environmental change (Daly-Engel et al. 2010).
Genetic Structure
In spite of their low levels of polymorphism and shallow
divergence, mtDNA sequences revealed strong genetic heterogeneity among samples collected in the NMP. Separate
and concatenated haplotypes supported strong genetic
structuring ( Φ s t -N D 2 = 0 .16 , P < 0.0001; Φ s t -C R = 0 .4 2,
P < 0.0001; and Φ s t -C A T = 0 .23 , P < 0.0001). Very little is
known about the genetic structure of batoid populations in
the NMP. The mtDNA of R. productus CR (Sandoval-Castillo
et al. 2004), R. steindachneri ND2 (Sandoval-Castillo and
Rocha-Olivares 2011), and G. marmorata Cyt-b (Smith et al.
2009) also exhibited strong genetic differentiation between
the PCBC and the GC, similar to the present investigation.
97
Journal of Heredity 
On the other hand, microsatellite variation in Urobatis halleri
showed that samples collected in southern California and the
central Gulf of California come from a panmictic population
(Plank et al. 2010).
Genetic differentiation may result from limited historical
or contemporaneous connectivity between regions produced
by geological events, oceanographic barriers, or reproductive behavior, which are factors known to play an important
role in the population structure of elasmobranchs (Dudgeon
et al. 2012; Portnoy and Heist 2012). Biogeographic events
that have influenced the evolutionary history of marine
populations in Baja California may explain the genetic discontinuity found in the mitochondrial DNA of the banded
guitarfish (Bernardi et al. 2003; Riginos 2005). The biogeographic history in the Baja California peninsula features a set
of important vicariant events. As a result of plate-boundary
expansion between the North American and Pacific plates,
the Baja California peninsula began to separate from the
Mexican mainland in the late Miocene, leading to the inception of the GC. Later, transpeninsular seaways were created
and connected the GC with the Pacific Ocean again (Riddle
et al. 2000), whose subsequent closure produced vicariant events in marine populations that have been postulated
to account for the patterns of genetic variation of several
species of fish in the NMP (Bernardi et al. 2003), including
batoids such as R. productus (Sandoval-Castillo et al. 2004) and
G. marmorata (Smith et al. 2009).
Abiotic factors are also likely to play an important role in
the genetic structuring of populations. The coastal habitat
of the west coast of the Baja California peninsula influenced
by the California Currents System is drastically different
from the conditions prevailing in the Gulf of California
in physical, chemical, and biological attributes (Lluch-Cota
et al. 2007; Suárez-Moo et al. 2013). Of particular relevance
may be the different thermal regimes in both regions, in
which the Gulf of California reaches considerably warmer
temperatures than the coastal water off the western Baja
California shores (Soto-Mardones et al. 1999). Habitat differences may cause an ecological oceanographic barrier in
the banded guitarfish. A similar pattern has been observed in
D. batis, in which surface water temperature has been found
to be a potential barrier limiting northerly and southerly distributions of clades off the British Isles in the north Atlantic
(Griffiths et al. 2010).
Patterns of reproductive behavior may also contribute
to the observed genetic differentiation. Female reproductive
philopatry to nursery areas has been documented in several
viviparous elasmobranchs (Dudgeon et al. 2009, 2012) including a few batoids (Chevolot et al. 2006b, 2007). Fidelity to
nursery areas prevents mature individuals from visiting new
areas for reproduction, reducing the possibility of genetic
exchange among reproductive areas. Zapteryx exasperata massive reproductive aggregations have been documented both
in Bahia Kino (GC), Bahía Vizcaíno, and Bahía Almejas
(PCBC) in late spring and early summer (VillavicencioGarayzar 1995; Blanco-Parra et al. 2009a; Cartamil et al.
2011). However, no mark–recapture or similar data are available regarding whether rays return to breed to the same areas
98
during each reproductive season and how many of them are
capable of using both reproductive areas inside and outside
the GC. Tagging studies are required to directly assess the
hypothesis of reproductive philopatry.
Finally, morphometric differences and contrasting patterns
of reproduction in GC and PCBC populations of banded guitarfish are phenotypic manifestations of population-level differences that correlate with the genetic differentiation found
in this study. Significant differences (P < 0.05) in mean total
length (TL) are found in rays caught in Bahia Vizcaino (PCBC:
TLmale = 79.9 ± 6.9 cm and TLfemale = 83.4 ± 10 cm) and in Sonora
(GC: TLmale = 66.06 ± 7.57 cm, TLfemale = 73.46 ± 10.42 cm)
(Blanco-Parra et al. 2009a; Cartamil et al. 2011). In addition,
populations show contrasting reproductive patterns; in Sonora
(GC), the ovarian cycle and gestation period occur simultaneously and females undergo embryonic diapause (BlancoParra et al. 2009b). Embryonic diapause is a temporary delay
in development at any stage of embryogenesis, it allows the
time between fertilization and parturition to be prolonged, and
species can benefit differently from this reproductive strategy
by delaying parturition until a time when newborns have the
most favorable conditions for survival or by allowing the synchronization of reproductive events (Waltrick et al. 2012). In
contrast, in rays from Bahía Almejas (PCBC), the ovarian cycle
is not concurrent with the gestation period, and pups are born
the same season they are conceived (Villavicencio-Garayzar
1995; Blanco-Parra et al. 2009b).
The regional genetic differentiation found in the mtDNA
of the banded guitarfish warrants extending our survey both
genetically and geographically to corroborate the extent of
the reproductive isolation signal in the genome of the species
and to potentially discover additional heterogeneity. Our data
are consistent with the existence of more than 1 reproductively isolated population of Z. exasperata in the NMP and
add to the increasing evidence attesting to the diversifying
nature of this evolutionarily dynamic region.
Management and Conservation Implications
The adoption of genetic and evolutionary criteria in the conservation and management of natural resources has led to the
recognition of “Management Units” (MUs), which represent
functionally independent populations or group of populations characterized by low levels of gene flow (Moritz 1994).
Accordingly, given the consistent patterns from different lines
of evidence, we propose to consider the existence of 2 MUs
for Z. exasperata in the NMP: one in GC (mainly in Sonora)
and other in the PCBC. Each MU possesses unique genetic and
phenotypic traits and therefore should be treated differently
and separately. Differences in life history must be considered
for the implementation of successful conservation and management plans given their potential relevance in providing resilience in the face of fishing mortality.
Supplementary Material
Supplementary material can be found at http://www.jhered.
oxfordjournals.org/.
Castillo-Páez et al. • Strong Population Structure in Banded Guitarfish
Funding
Centro de Investigación Científica y de Education Superior de
Ensenada (no. 625112) grant to A.R.O.; a postgraduate scholarship (no. 237074) from the Consejo Nacional de Ciencia y
Tecnología (CONACYT) provided support to A.C.P. during her
M.Sc. program in Marine Ecology at Centro de Investigación
Científica y de Education Superior de Ensenada.
Acknowledgments
We thank Erick Oñate and Omar Santana from the Fisheries Ecology
Laboratory (CICESE) for their support during fieldwork in PCBC and
José Domínguez for helping with figure preparation. We thank Instituto
Politecnico Nacional for awarded fellowships to F.G.M. We are grateful to 3
anonymous reviewers for their constructive comments.
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Received April 7, 2013; First decision June 11, 2013;
Accepted September 4, 2013
Corresponding Editor: Jose Lopez