Mitochondrial DNA Part A

DNA Mapping, Sequencing, and Analysis

ISSN: 2470-1394 (Print) 2470-1408 (Online) Journal homepage: http://www.tandfonline.com/loi/imdn21

Proposed synonymy for Micropogonias altipinnis

(Gnther 1864), Micropogonias ectenes (Jordan
& Gilbert 1882), and Micropogonias megalops
(Gilbert 1890)

Geremas Snchez-Pinedo, No Daz-Viloria, Jos L. Ortiz-Galindo, Nelson

Ferreira-Fontoura, Ricardo Perez-Enriquez, Laura Snchez-Velasco & Jos De
La Cruz-Agero

To cite this article: Geremas Snchez-Pinedo, No Daz-Viloria, Jos L. Ortiz-Galindo, Nelson

Ferreira-Fontoura, Ricardo Perez-Enriquez, Laura Snchez-Velasco & Jos De La Cruz-Agero
(2017): Proposed synonymy for Micropogonias altipinnis (Gnther 1864), Micropogonias
ectenes (Jordan & Gilbert 1882), and Micropogonias megalops (Gilbert 1890), Mitochondrial
DNA Part A, DOI: 10.1080/24701394.2016.1258405

To link to this article: http://dx.doi.org/10.1080/24701394.2016.1258405

Published online: 24 Jan 2017.

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MITOCHONDRIAL DNA PART A, 2017
http://dx.doi.org/10.1080/24701394.2016.1258405

RESEARCH ARTICLE

Proposed synonymy for Micropogonias altipinnis (Gu nther 1864), Micropogonias

ectenes (Jordan & Gilbert 1882), and Micropogonias megalops (Gilbert 1890)
Geremas Sanchez-Pinedoa, Noe Daz-Viloriaa, Jose L. Ortiz-Galindoa, Nelson Ferreira-Fontourab,
Ricardo Perez-Enriquezc, Laura Sanchez-Velascoa and Jose De La Cruz-Agu eroa
a
Instituto Politecnico NacionalCentro Interdisciplinario de Ciencias Marinas (IPN-CICIMAR), La Paz, B.C.S, Mexico; bFaculdade de
Bioci^enciasPontifcia Universidade Catolica do Rio Grande do Sul (FaBio-PUCRS), Porto Alegre, RS, Brazil; cCentro de Investigaciones
Biologicas del Noroeste (CIBNOR), La Paz, B.C.S, Mexico

ABSTRACT ARTICLE HISTORY

Within the Sciaenidae family, the genus Micropogonias is composed of three recognized species along Received 15 September 2016
the Pacific coast of Mexico: Micropogonias altipinnis, M. ectenes, and M. megalops. These species exhibit Accepted 4 November 2016
overlapping diagnostic characters, which make species identification difficult. This study ties morpho-
logical differences (meristic, morphometry of body, and otolith) with DNA sequences (CO1 and 16S frac-
KEYWORDS
tions of mtDNA and 28S of nDNA) among Micropogonias species in the Pacific. Meristic analysis showed CO1; morphological and
a latitudinal variation among the three species in the number of rays, the number of gill rakers, and molecular characters;
length of the longest spine of the dorsal fin. Discriminant analysis of morphometric characters (body Micropogonias megalops;
and otolith) showed three morphological entities (p < 0.001). However, the mean genetic divergences Micropogonias ectenes;
among the three species with partial sequences of mtDNA (CO1 and 16S), and nuclear (28S) were lower Micropogonias altipinnis;
than those reported at the interspecific level (>2%). Genetic results suggest that the three species are tropical eastern Pacific
one species and that the differences in meristics and morphometry could be the result of phenotypic
plasticity or incipient speciation. In this sense, M. ectenes and M. megalops are proposed as junior syno-
nyms of M. altipinnis.

Introduction
The Sciaenidae fish family (commonly called drums or
croakers) is composed of 24 genera (Chao 1995) in the trop-
ical eastern Pacific (TEP), a region that stretches from the Gulf
of California to northern Peru (Briggs 1974). In the TEP, two
biogeographic provinces have been defined, the Cortez
Province (Gulf of California and lower Pacific Baja California
Peninsula) and the Panamic Province (south of the TEP),
based on faunal complexes, regional endemics, and three
functional groups of shore fish species (reef, soft-bottom, and
coastal pelagic). This reflects speciation within each province
(Robertson & Cramer 2009). Among the provinces, the genus
Micropogonias is represented by three valid species: M. alti-
pinnis (Gunther 1864), M. ectenes (Jordan & Gilbert 1882), and
M. megalops (Gilbert 1890). These three species share, to
some extent, their geographic range (Chao 1995). The slender
croaker (M. ectenes) ranges from the southern Gulf of
California and the southwestern coast of the Baja California
Peninsula to Oaxaca, Mexico. The tallfin croaker (M. altipinnis)
ranges throughout the Gulf of California and the tip of the
Baja California Peninsula to Peru. The bigeye croaker (M. meg-
alops) ranges throughout the Gulf of California to Oaxaca,
Mexico (Figure 1).
In general, the identification of fish species is based on
external morphological features, including body shape, colour
pattern, scale size and count, the relative position of fins,
number and type of fin rays, the number of gill rakers, and/or
various relative measurements of body parts. Occasionally,
otoliths and other specific bones are used for species discrim-
ination (Teletchea 2009).
Identification of Micropogonias species in the TEP (Chao
1995), based on distinctive characteristics, is difficult because
they are very similar. Original descriptions of the three
Micropogonias species were based on a few individuals col-
lected at three locations; two specimens of M. altipinnis in
Panama and Guatemala, four of M. ectenes at Sinaloa, Mexico,
and four of M. megalops at Sonora, Mexico. These and other
difficulties explain why researchers have attempted to develop
new methods for identifying fish species without relying only
on morphological features (Teletchea 2009).
Identification based on genetics compares sequences of
genes, usually mtDNA loci that are known to vary between
species. Mitochondrial genes have a high copy number per
cell, typically lack recombination (promoting the loss or fix-
ation of mtDNA haplotypes), reducing diversity within spe-
cies, and enabling species identification (Dawnay et al. 2007).
Additional mtDNA features include patterns of maternal
inheritance and rapid rates of evolutionary change in com-
parison to nuclear DNA, making it a suitable tool for genetic
studies among taxa of several fish groups (Turan 2008). DNA
barcoding has shown that the mitochondrial DNA cyto-
chrome c oxidase subunit 1 gene (CO1) can be used to

CONTACT Noe Daz-Viloria ndviloria@hotmail.com Instituto Politecnico Nacional Centro Interdisciplinario de Ciencias Marinas (IPN-CICIMAR), Avenida IPN
s/n, La Paz, B.C.S. 23096, Mexico
2017 Informa UK Limited, trading as Taylor & Francis Group
2 
G. SANCHEZ-PINEDO ET AL.
Figure 1. Range of M. altipinnis (black continuous line), M. ectenes (black segmented line), and M. megalops (grey continuous line) along the Pacific coast of Mexico.
Sample sites of Micropogonias species. 1: M. megalops at San Felipe, Baja California; 2 and 3: M. ectenes at La Paz, Baja California Sur, and Mazatlan, Sinaloa (respect-
ively); 4: M. altipinnis at Salina Cruz, Oaxaca.
differentiate most species (Hebert et al. 2003; Ward et al.
2005; Kim et al. 2010).
Recent studies based on morphological data and sequen-
ces of mtDNA have suggested a single taxonomic entity in
some fish previously considered different species. This is the
case of hake (Merluccius; Silva-Segundo et al. 2011), gadoids
(Theragra; Byrkjedal et al. 2008), and gerreids (Eugerres;
Martnez-Guevara et al. 2015). MtDNA is also used for identi-
fying cryptic species (Gambusia; Lara et al. 2010; Albula gil-
berti and A. esuncula; Pfeiler et al. 2011), and Indian and
western Pacific types of Lutjanus russellii into two species
(Chu et al. 2013).
If the TEP contains three species of Micropogonias with
some sympatry, then morphological data and DNA sequences
should differentiate them, supporting the current taxonomic
classification of Micropogonias species. This study compares
morphological differences (meristic, morphometry of body,
and otoliths) with DNA sequences (CO1 and 16S fractions of
mtDNA and 28S of nDNA) among M. altipinnis, M. ectenes,
and M. megalops.
Materials and methods
Sampling
We collected 125 specimens of the three Micropogonias spe-
cies at four locations along the coast of the TEP from
September 2012 through June 2013: 43 specimens of M. meg-
alops at San Felipe, Baja California (31
010 N, 114
490 W); 31
specimens of M. ectenes at La Paz, Baja California Sur (24
090 N,
110
190 W) and six at Mazatlan, Sinaloa (23
120 N, 106
250 W);
and 45 specimens of M. altipinnis at Salina Cruz, Oaxaca
(16
100 N, 95
100 W) (Figure 1). Fish were collected by fishermen
with gill nets at the four locations, frozen, and transported on
ice to IPNCICIMAR for processing. Some specimens were
deposited in the fish collection of IPNCICIMAR (http://colec-
cion.cicimar.ipn.mx).
Morphological analysis
Identification was based on keys (Chao 1995) and original
diagnoses (Gu nther 1864; Jordan & Gilbert 1882; Gilbert 1890;
Jordan & Evermann 18961900). The number of gill rakers,
barbels, and rays of the dorsal fin were counted. The position
of the longest spine in relation to the first two rays of the
dorsal fin, when the dorsal fin was folded towards the body,
was taken into account. We made 18 morphometric measure-
ments of each specimen (Figure 2), using a vernier caliper at
small measurements, and a measuring ruler or measuring
tape at bigger measurements. The measurements were made
as follows: (1) Head length from the tip of the snout to the
beginning of the operculum, (2) Pectoral fin length from the
tip to the base of the fin, (3) Predorsal length from the tip of
the snout to the beginning of the dorsal fin, (4) Postdorsal
length from the beginning dorsal fin to the beginning of the
dorsal rays of caudal fin, (5) Interocular length measured as
the distance between eyes, (6) Caudal fin length from the tip
of the fin to the base of rays, (7) Horizontal eye diameter
from the anterior to the posterior edge of eye, (8) Head
height from the operculum isthmus to the dorsal edge of the
head, (9) Pelvic fin length from the tip of the fin to the base
of rays, (10) Preorbital length from the tip of snout to the
anterior edge of eye, (11) Maximum body height, (12) Anal
fin length from the beginning to the end of the fin, (13)
Caudal peduncle length from the end of the anal fin to the

Figure 2. Morphometric measurements. 1: Head length; 2: pectoral fin length; 3: predorsal length; 4: postdorsal length; 5: interocular length; 6: caudal fin length; 7:
horizontal eye diameter; 8: head height; 9: pelvic fin length; 10: preorbital length; 11: maximum body height; 12: anal fin length; 13: caudal peduncle length; 14:
spine of dorsal fin length; 15: dorsal fin length; 16: postorbital length; 17: preanal length; 18: standard length.
beginning of the ventral rays of caudal fin, (14) Spine of dorsal
fin length from the base to the tip of the largest spine, (15)
Dorsal fin length from the beginning to the end of the fin, (16)
Postorbital length from the posterior edge of the eye to the
edge of operculum, (17) Preanal length from the tip of the snout
to the beginning of the anal fin, and (18) Standard length from
the tip of the snout to the beginning of the caudal-fin rays.
All measurements were transformed to avoid bias caused
by the size of specimens, using the equation: Ms Mo (Ls/Lo)b,
where b regression Log10 Mo in Log10 Lo, Ms is the standar-
dized measurement, Mo is the measurement to be standar-
dized, Ls is the standard length of all fish, and Lo is the
measurement to standardize all fish (Elliott et al. 1995). A dis-
criminant analysis of body measurements was performed to
assess patterns of variation and identify morphological char-
acters that separate two or more groups or putative species.
Sagittal otoliths of every fish were removed, cleaned, and
photographed under a stereomicroscope with an attached
camera. Pictures of the internal proximal side were taken and
standardized scale was used as a reference. Measurements on
the otolith pictures were done relative to sulcus, using
Image-Pro Plus 6.0 software. The sulcus was divided into the
ostium and cauda, the division was at the crista (Figure 3).
Six measurements obtained from the automated menu of the
software and 21 ratios of lengths and total area were com-
pared for the three species by discriminant analysis.
Genetic analysis
We prepared 25 samples of muscle tissue: M. altipinnis
(n 10), M. ectenes (n 10), and M. megalops (n 5).
Genomic DNA extractions were carried out following a modi-
fied protocol of Lopera-Barrero et al. (2008). Prior to PCR,
DNA concentrations of samples were standardized to
50100 ng lL1. The partial sequences of mtDNA genes: CO1
(700 bp) and 16S rRNA (600 bp), and nDNA 28S (847 bp) were
amplified by PCR. The CO1 fraction was amplified with pri-
mers: VF1 (50 -TTCTCAACCAACCACAAAGACATTGG-30 ) (Ivanova
et al. 2007) and FishR1 (50 -TAGACTTCTGGGTGGCCAAAG
AATCA-30 ) (Ward et al. 2005). 16S rRNA (16S) was amplified
with 16Sa (50 -CGCCTGTTTAACAAAAACAT-30 ) and 16Sb (50 -
CCGGTTTGAACTCAGATCACGT-30 ) (Palumbi 1996). 28S was
MITOCHONDRIAL DNA PART A 3
Figure 3. Left sagitta otolith of Micropogonias specimen and its parts: 1: sulcus;
2: ostium; 3: cauda; 4: crista. The position of the otolith in reference to spec-
imens body: D: dorsal; V: ventral; A: anterior; P: posterior.
amplified with C1 (50 -ACCCGCTGAATTTAAGCAT-30 ) and C2 (50 -
TGAACTCTCTCTTCAAAGTTCTTTTC-30 ) (Le^ et al. 1993). DNA
amplifications were performed in a PCR thermal cycler under
the following conditions: 2 min at 94
C, 35 cycles of 1 min at
94
C, 1 min at 50
C (CO1), 53
C (16S), or 42
C (28S), 1 min
at 72
C, and a final extension of 4 min at 72
C. PCR products
(20 lL, 100 ng lL1) were sequenced in sense and antisense
directions in an automatic sequencer (ABI Prism 3730XL,
Applied Biosystems, Carlsbad, CA) at Macrogen (Seoul, South
Korea).
CO1 and 16S sequences of three Micropogonias species
from the GenBank were used as references (Table 1). The
best model of nucleotide substitution was selected with
MEGA 6.0 software (Tamura et al. 2013) for the construction
of the maximum-likelihood tree. Genetic distances of K2P G
(Kimura-two-parameter Gama) among reference sequences
of CO1 and 16S fractions and sequences of M. altipinnis, M.
ectenes, and M. megalops were used to display phylogenetic
relationships by the maximum-likelihood tree (5000 boot-
straps), using MEGA 6.0 software (Tamura et al. 2013).
Haplotype frequencies of CO1 were obtained for each
Micropogonias species from the TEP and M. furnieri and M.
undulatus from the Atlantic, using DnaSP 5.10 (Librado &
Rozas 2009). A maximum parsimony haplotype network,
using the Network 5 genetics software (Bandelt et al. 1999;
Polzin & Daneschmand 2003) was obtained with the CO1
4 
G. SANCHEZ-PINEDO ET AL.

sequences, using M. furnieri as the outgroup. Time of differen-
tiation between haplotypes was estimated, using two
molecular clocks: 1.3% and 2% per million years (Lessios
2008; DiBattista et al. 2013), using the time estimate subpro-
gram of Network 5 (Forster et al. 1996; Saillard et al. 2000).
of otoliths, identified a priori, showed significant differences
(F 16.048, Wilk's c 0.0089, p < .0001), similar to body
measurement analyses. Otoliths of M. altipinnis were different
from those of M. ectenes and M. megalops; however, some
otoliths of M. ectenes were classified with M. altipinnis and M.
megalops forms (Figure 5).

Results
Morphological analysis
The number of gill rakers in the first-gill arch ranged from
1417 in M. megalops, 1216 in M. ectenes, 1315 in M. alti-
pinnis. The ranges overlapped. The number of dorsal fin rays
showed a range from 2628 in M. megalops, 2427 in M.
ectenes, and 2123 in M. altipinnis. These ranges showed dif-
ferences between M. megalops and M. altipinnis and between
M. altipinnis and M. ectenes, but M. ectenes and M. megalops
overlap. Among the three species, all had four pairs of bar-
bels. The longest spine reaching the first two rays of the dor-
sal fin when the dorsal fin was folded towards the body were
none of the M. megalops, five of the 37 M. ectenes, and 38 of
the 40 M. altipinnis.
Discriminant analysis with 17 morphometric variables
(Table 2) showed three different morphological entities
(F 34.212, Wilks c 0.002, p < .000). The dorsal fin length,
longest spine length of the dorsal fin, and the preanal length
were among the five most discriminant variables (Table 2). In
this analysis, M. altipinnis and M. megalops were different;
M. ectenes showed an intermediate morphotype, and
M. altipinnis was the most distinctive species (Figure 4).
Of 27 variables of otoliths, 11 had differences; 4 were
automatically detected by the software and 7 were length
and total area ratios (Table 3). Discriminant analysis in groups
Genetic analysis
We obtained 25 sequences of CO1 (572 bp), 25 of 16S
(543 bp), and 25 of 28S (277 bp) for the three species (M.
megalops, M. ectenes, and M. altipinnis). All sequences were
deposited in GenBank (Accession numbers: KX401587
KX401611, KX417648KX417697).
The fraction of the mtDNA CO1 region of Micropogonias
species from the TEP (10 individuals each), and Atlantic spe-
cies (five and four individuals of M. furnieri and M. undulatus,
respectively) revealed 23 haplotypes. Four haplotypes were
shared between M. altipinnis and M. ectenes and four haplo-
types were unique to either species. Six haplotypes, including
the most frequent, were unique to M. megalops. M. furnieri,
and M. undulatus, revealing three and two unique haplotypes,
respectively (Table 4).
Intraspecific genetic divergences for CO1 ranged from
01.06% in M. megalops and 00.88% in M. ectenes and M. alti-
pinnis. In Atlantic species, divergences ranged from 00.17% in
M. undulatus and 00.53% in M. furnieri. Interspecific genetic
divergences from the TEP were low. Divergences between M.
ectenes and M. megalops ranged from 0.171.42%, between M.
altipinnis and M. ectenes from 01.24%, and between M. altipin-
nis and M. megalops from 0.351.78%. Comparisons among
Atlantic and TEP species showed divergences that ranged from
2.68%7.30%. Atlantic species (M. furnieri and M. undulatus)

Table 1. Reference sequences of Micropogonias species of TEP (M. megalops) and Atlantic (M. furnieri and M. undulatus), with their GenBank accession
numbers. n, number of sequences per species.
CO1 16S
Micropogonias species n Accession No. n Accession No.
M. megalopsa 5 KC208689KC208693 5 KC208666KC208670
M. furnieri 5 GU702481, GU702432, GU702483, GU702484, GU702534 1 AY603006
M. undulatus 4 JQ841936, JQ841938, JN021309, KC015693 1 FJ175392
a
Individuals of M. megalops were identified by the same research team, using the same identification keys.

Table 2. Morphometric measurements of body parts used in the discriminant analysis of M. altipinnis, M. ectenes, and M. megalops.
Length Variables Matrix % F Wilks c Root 1 Root 2 p
Head 17 100 34.21 0.0029 99.30 0.60 .00
Pectoral-fin 16 100 32.21 0.0030 99.39 0.60 .00
Predorsal 15 100 30.21 0.0031 99.39 0.60 .00
Postdorsal 14 100 28.21 0.0032 99.38 0.61 .00
Interocular 13 100 26.22 0.0034 99.41 0.58 .00
Caudal fin 12 100 24.22 0.0036 99.39 0.60 .00
Horizontal eye diameter 11 100 22.22 0.0038 99.38 0.61 .00
Head height 10 100 20.22 0.0040 99.45 0.54 .00
Pelvic fin 9 99.2 18.22 0.0043 99.49 0.50 .00
Preorbital 8 100 16.23 0.0045 99.47 0.52 .00
Maximum body height 7 100 14.23 0.0048 99.55 0.44 .00
Anal fin 6 99.2 12.23 0.0052 99.63 0.36 .00
Caudal peduncle 5 98.4 10.23 0.0058 99.75 0.24 .00
Spine of the dorsal fin 4 98.4 8.23 0.0064 99.84 0.15 .00
Dorsal fin 3 98.4 6.24 0.0076 99.93 0.06 .00
Postorbital 2 97.6 4.24 0.0084 99.94 0.05 .00
Preanal
In bold the most discriminant variables.
 MITOCHONDRIAL DNA PART A 5

Figure 4. Scatterplot of classification functions, based on 17 morphometric variables. The first axis (Root 1) explained 98.88% of the variation and the second axis
(Root 2) explained 1.12%. M. altipinnis in grey squares, M. ectenes in black circles, and M. megalops in black triangles.

Table 3. Significant measurements and ratios of otoliths used in the discriminant analysis of M. altipinnis, M. ectenes, and M. megalops.
Length and ratios Wilk's c F p
Fouriers description of otolith 0.458 50.395 < .0001
Maximum diameter of cauda (mm)/maximum diameter of otolith (mm) 0.690 19.136 < .0001
Medium diameter of cauda (mm)/medium diameter of otolith (mm) 0.797 10.804 < .0001
Minimum diameter of ostium (mm)/minimum diameter of otolith (mm) 0.803 10.412 < .0001
Eclipticity of otolith 0.206 163.359 < .0001
Eclipticity of ostium 0.231 141.397 < .0001
Eclipticity of cauda 0.212 158.047 < .0001
Maximum length of cauda (mm)/maximum length of otolith (mm) 0.772 12.560 < .0001
Area of cauda (mm2)/area of otolith(mm2) 0.810 9.981 .000
Polygonal area of ostium (mm2)/Polygonal area of otolith (mm2) 0.832 8.600 .000
Medium diameter of ostium (mm)/Medium diameter of otolith (mm) 0.961 1.730 .184

had the most divergence, from 6.887.28% (Table 5). Using 16S
and 28S sequences, the highest interspecific divergence among
the TEP and Atlantic species was 1.6% and 0%, respectively.
The maximum-likelihood tree, using the CO1 sequence
(Figure 6), groups M. ectenes, M. megalops, and M. altipinnis
in the same clade, with a 69% of bootstrap. Atlantic species
were grouped in different clades with high bootstrap percen-
tages. Micropogonias furnieri was grouped with 99% boot-
strap and M. undulatus with 100% bootstrap. Within the clade
of species from the TEP, three groups with low bootstrap per-
centages (less than 50%) were present. At the top was a
group with M. ectenes and M. altipinnis. In the middle was
a group of M. ectenes and M. megalops. At the bottom was
a group with M. ectenes and M. altipinnis. While M. altipinnis
and M. megalops were not together in any clade, M. ectenes
was present in three groups with the other two species
(Figure 6).
The maximum-likelihood tree, using 16S, showed one
clade containing all TEP species, including the sequence of
M. furnieri (AY603006). Only the sequence of M. undulatus
was outside this clade (figure not shown). The maximum-
likelihood tree, using 28S, was not constructed because no
variations were observed in this fraction.
The parsimony network (Figure 7) revealed that
Micropogonias haplotypes from the TEP (H2, H6, H11, and
H13) had the fewest mutation steps (1719 mutations) com-
pared with haplotype 19 (H19) of M. furnieri, than any haplo-
types of M. undulatus (3536 mutations, figure not shown).
Four haplotypes (H2, H4, H6, and H8) were shared by M. alti-
pinnis and M. ectenes. Haplotypes were not shared by M.
megalops and M. ectenes; however, three haplotypes of M.
ectenes (H9, H11, and H12) were connected to M. megalops
haplotypes by singletons and connected to shared haplo-
types of M. altipinnisM. ectenes (H2 and H6) by five mutation
steps (0.87% of genetic divergence). Central haplotypes of M.
altipinnisM. ectenes and M. megalops with higher frequencies
(H2 and H13, respectively) were different only by three muta-
tional steps (0.52% of genetic divergence).
Since some haplotypes were shared or were different by
one mutation step, we decided to group some haplotypes
and named as follows: lineages M. altipinnisM. ectenes
(M.a.M.e.) and lineages M. megalopsM. ectenes (M.m.M.e.).
6 
G. SANCHEZ-PINEDO ET AL.
Figure 5. Scatterplot of classification functions based on 11 otoliths variables. The first axis (Root 1) explained 86.28% of the variation and the second axis (Root 2)
explained 13.72%. M. altipinnis in gray squares, M. ectenes in black circles, and M. megalops in black triangles.
The former were found in the southern TEP (Panamic
Province) and the later found in the northern TEP (Cortez
Province) (Figure 7, Table 6).
Estimated divergence time among Micropogonias furnieri
and M. altipinnisM. ectenes and M. megalopsM. ectenes line-
ages ranged from 1.2 mya to 1.9 mya. Estimated divergence
time between M. altipinnisM. ectenes and M. megalopsM.
ectenes lineages ranged from 218 kya to 336 kya (Table 6).
Discussion
Morphological analysis
Until now, differentiation of species of Micropogonias used
meristics and the position of the longest spine of the dorsal
fin (Chao 1995). We found that meristics revealed latitudinal
variation in the gill rakers, rays of the dorsal fin, and the
number of fish with the longest spine reaching the first two
rays of the dorsal fin.
Latitudinal variation in meristic characteristics, potentially
associated with environmental and ocean-climate variations,
have been documented in Micropogonias and other fish
(Lindsey 1954; Vazzoler 1971; Figueroa & Daz de Astarloa
1991; Silva-Segundo et al. 2011). In M. furnieri, differences in
the number of gill rakers, lateral line scales, and/or fin rays
were reported between northern and southern populations
of Brazil (Vazzoler 1971) and Uruguay (Figueroa & Daz de
Astarloa 1991). These types of latitudinal variations have also
been reported in the paradise gourami Macropodus opercula-
ris (Lindsey 1954), and the Pacific hake Merluccius spp.
(Silva-Segundo et al. 2011). Earlier, controlled experiments in
salmonids showed that the variation in environmental param-
eters (temperature, oxygen) could modify the number of
vertebrae or dorsal and anal fin rays (Seymour 1956; Orska
1957). Thus, based on this and on current genetic informa-
tion, meristic characteristics cannot be the only approach to
establish taxonomic status of a species.
Genetic analysis
Avise (2000) and Hebert et al. (2003) suggest that interspe-
cific genetic variation is higher than 2% and intraspecific is
less than 1%. Our results contrast with these ranges, as
Micropogonias interspecific divergences in the TEP were <2%,
with some even lower than intraspecific ones (Table 5).
Nevertheless, a comparison of CO1 sequences of 22
Sciaenidae species in the TEP (Table 7) and Micropogonias
species in the TEP and Atlantic indicate that interspecific
divergence ranges from 2.6819.29% and the range of intra-
specific divergence is 01.2% (Figure 8). These results suggest
that Micropogonias species from the TEP conform to a single
species rather than three separate species.
The maximum-likelihood tree indicated significant differen-
ces between Atlantic and TEP species, but no significant dif-
ferences among Micropogonias in the TEP, supported these
observations, notwithstanding that M. altipinnis and M. mega-
lops did not share haplotypes. Silva-Segundo et al. (2011)
report similar low genetic differences (0.40.8%) among three
hake Merluccius species. They suggest that these should not
be considered separate species because there was no mono-
phyletic clade, as defined by the phylogenetic species con-
cept (De Queiroz 2007). Rather, the tree reflects two
population units with some degree of gene flow. Similarly,
Byrkjedal et al. (2008) found non-significant differences for
CO1 between Theragra finnmarchica and T. chalcograma,
Table 4. Frequencies and variable sites of each haplotype (H) of the CO1 fraction of 572 bp of M. altipinnis (Ma), M. ectenes (Me), M. megalops (Mm), M. furnieri (Mf), M. undulatus (Mu).
Variable sites
H Ma Me Mm Mf Mu
Freq 3 23 26 77 95 98 110 131 137 140 152 164 170 182 188 191 203 206 209 230 233 248 251 269 275 290 302 305 308
H13 5 G G G C G A T G C A C T T A C T T G G T T G A A G A C C C
H2 1 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
H8 1 2 . . . . . . . . . . . . . . . . . . . . . A . . . . . . .
H4 1 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
H5 2 . . . . . . . . . . . G . . . . . A . . . . . . . . . . .
H6 1 1 . . . . . . . . . . . . C . . . . . . . . . . . . . . . .
H7 2 . . . . . . . . . . . . C . . . . . . . . . . . . . . . .
H1 1 . . . . A . . . . . . . . . . . . . . . . . . . . . . . .
H3 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
H9 1 . . . . . . . A . . . . . . . . . . . . . . . . . . . . .
H10 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
H11 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
H12 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
H14 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
H15 1 . . . . . . . . . . . . . . . . . A . . . . . . . . . . .
H16 1 . . . . . . . . . . . . . . . C . . . . . . . . . . . . .
H17 1 . . . . . . . . . . . . . . . . . . . . . . . . . . T . .
H18 1 . . . . . . . . . . . . . . . C . . . . . . . . . . . T .
H19 3 A A . . . G T . . . . . . . . . . . . . C . . . . . . . T
H20 1 A A . . . G . . . . . . . . . . . . . . C . G . . . . . T
H21 1 A A . . . G . . . . . . . . . . . . . . C . . . . . . . T
H22 3 A A A T . . C . T G T . C G T C C A A C C . G G A G . T .
H23 1 A A A T . . C . T G T . C G T C . A A C C . G G A G . T .
Freq 311 317 320 332 338 353 356 362 383 401 404 410 413 428 452 455 464 467 482 497 498 506 509 515 521 527 539 542 563
H13 5 A C G C G G C C C T C T T C G T T G T A C A C T A T A G C
H2 1 2 . . . T . . . . T . . . . . . . . . . . . . . . . . . . .
H8 1 2 . . . T . . . . T . . . . . . . . . . . . . . . . . . . .
H4 1 1 . . . T . . T . T . . . . . . . . . . . . . . . . . . . .
H5 2 . . . T . . . . T . . . . . . . . . . . T . . . . . . . .
H6 1 1 . . . . . . . . T . . . . . . . . . . . . . . . . . . . .
H7 2 . . . T . . . . T . . . . T . . . . . . . . . . . . . . .
H1 1 . . . T . . . . T . . . . . . . . . . . . . . . . . . . .
H3 1 . . . T . . . . T . . . . . . C . . . . . . . . . . . . .
H9 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
H10 1 . T . T . . . . T . . . . . . . . . . . . . . . . . . . .
H11 1 . . . . A . . . . . . . . . . . . . . . . . . . . . . . .
H12 1 . . . . . . . . . C . . . T . . . . . . . . . . . . . . .
H14 1 . . . . . . . . . . . . . T . . . . . . . . . . . . . . .
H15 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
H16 1 . . A . . . . . . . . . . . . . . . . . . . . . . . . . .
H17 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
H18 1 . T A . . . . . . . . . . . . . . . . . . . . . . C . . .
H19 3 C . . . A A . . T . T . . . . . . A C C . . . . C C . . A
H20 1 C . . . A A . . T . T . . . . . . A C C . . . . C C . . A
H21 1 C . . . A A . . T . T . . . . . . A . C . . T . C C . . A
H22 3 . . A . . . . T A . T C C . A . C A . . . G . C C . G A T
H23 1 . . A . . . . T A . T C C . A . C A . . . G . C C . G A T
MITOCHONDRIAL DNA PART A

A double hyphen represents no sample. The number of variable sites representing the nucleotide and their positions in the sequence and a point represents the same nucleotide as in H13.
7
8 
G. SANCHEZ-PINEDO ET AL.

Table 5. Minimum and maximum values (between parentheses) and mean percentages of genetic divergences of CO1 mtDNA fraction, obtained with Kimura-
two-parameter (K2P) model, among Micropogonias species from TEP (M. altipinnis, M. ectenes, and M. megalops) and Atlantic (M. furnieri and M. undulatus).
Species M. altipinnis M. ectenes M. megalops M. furnieri M. undulatus
M. altipinnis (00.88) 0.51
M. ectenes (01.24) 0.52 (00.88) 0.48
M. megalops (0.351.78) 0.76 (0.171.42) 0.56 (01.06) 0.34
M. furnieri (2.863.60) 3.16 (2.683.42) 3.01 (2.863.60) 3.05 (00.53) 0.21
M. undulatus (6.507.30) 7.13 (6.507.30) 7.12 (6.317.10) 6.81 (6.887.28) 7.19 (00.17) 0.09

Figure 6. Maximum likelihood tree, based on the partial sequences of the CO1 (572 bp) gene in mitochondrial DNA. Numbers beside the branch indicate bootstrap
values (>50%), based on 5000 replicates. Accession number of new sequences (our study) are in bold. Scale bar represents the genetic distance of K2P G.

formerly considered as valid species, although they were same species. In this context, some authors have recom-
different in several phenotypic characters. mended synonymization of taxa with morphological similarity,
Templenton (1992) proposed that individuals of popula- based on low genetic differentiation of CO1 sequences (Carr
tions with sufficient genetic similarity belong to the et al. 1999; Byrkjedal et al. 2008; Martnez-Guevara et al. 2015),
 MITOCHONDRIAL DNA PART A 9

Figure 7. Network of haplotypes shared among sampling locations. In white, haplotypes of M. altipinnis morphotype (Salina Cruz, Oaxaca), in black haplotypes of M.
ectenes morphotype (La Paz, Baja California Sur and Mazatlan, Sinaloa), in dark grey M. megalops morphotype (San Felipe, Baja California), and with diagonal lines M.
furnieri (Atlantic). TEP: tropical eastern Pacific; H: Haplotype; mv (light grey), unsampled intermediate haplotypes. The number between haplotypes represent muta-
tion steps.

Table 6. Estimated time of differentiation between Micropogonias lineages Table 7. Sciaenidae species from TEP, used in intraspecific and interspecific
from TEP and Atlantic, obtained in years with two molecular clocks: 1.3% and (sister species) genetic-variation analyses, represented in Figure 8.
2% per million years (above and below of diagonal, respectively). Standard Species n GenBank accession number
deviations in years are between parentheses.
Atractoscion nobilis 8 EU547246, GU440241, JQ741165, KM019240,
M.a.M.e. M.m.M.e. M.f. KM019242KM0192244, KM077533
M.a.M.e. 336202 (159031) 1849114 (436578) Cheilotrema saturnum 3 EU547247, GU440274, KP722707
M.m.M.e. 218532 (103371) 1949974 (455849) Corvula macrops 2 KP722711, KP722712,
M.f. 1267488 (296303) 1201929 (283777) C. othonopterus 7 KC208685, KR632701KR632706
M.a.M.e.: Lineages of M. altipinnisM. ectenes (Haplotypes 18, 10); M.m.M.e.: C. parvipinnis 2 GU440301, KP722715,
Lineages of M. megalopsM. ectenes (Haplotypes 9, 1118); M.f.: Haplotype C. praedatorius 1 KP722716
19 of M. furnieri. C. reticulatus 14 JQ398423JQ398427, KC208671, KC208673,
KC208676KC208680, KR632713, KR632714
Genyonemus lineatus 17 JQ354103, JQ354104JQ354106, JQ934981,
highlighting that morphological data and complementary KM019241,KM019300KM019309, KP722722,
molecular approaches resolve complex taxonomic situations, Isopisthus remifer 5 KP722723, KX401612KX401615
where an unclear differentiation among species exists. M. elongatus 1 KC208687
M. nasus 8 KR632720KR632727
The genetic divergences and bootstrap percentages of M. undulatus 4 EU547249, GU440404, KF930123, KP722737
Micropogonias species from the TPE, as noted in this study, Ophioscion scierus 1 KP722750
statistically corroborated the presence of a single species. The O. vermicularis 1 KP722751
Roncador stearnsii 4 EU547248, GU440506, GU440507, KP722773
existence of shared haplotypes among specimens of different Seriphus politus 7 KJ433961, KM077527KM077530, KM077550,
regions suggests high gene flow throughout its range. KP722777
Unshared regional haplotypes suggest population subunits Stellifer ericymba 1 KP722778
S. oscitans 1 KP722780
(subpopulations), rather than separate species. Meristic, mor- Totoaba macdonaldi 5 KC208681KC208684, KP722782
phological, and morphometric evidence (body and otoliths) Umbrina bussingi 1 KP722783
suggested the presence of different regional morphotypes, U. roncador 2 GU440563, KP722786
U. xanti 1 KP722787
probably driven by environmental factors.
n: number of sequences per species. Accession number of new sequences (our
The maximum parsimony network indicates that old line- study) are in bold.
ages of M. altipinnisM. ectenes morphotypes (H2) and M. meg-
alops (H13) are similar in ancestry because of their
10 
G. SANCHEZ-PINEDO ET AL.

Figure 8. Intraspecific and interspecific genetic divergences in CO1 sequences of sciaenid species from the tropical eastern Pacific and Atlantic (M. furnieri and M.
undulatus). Minimum, maximum and mean values were obtained with the Kimura-2-parameter (K2P) model. Total number of comparisons: 17 intraspecific (among
individuals from the same species) and 24 interspecific (among individuals from 17 Sciaenidae sister species). An: Atractoscion nobilis; Cs: Cheilotrema saturnum; Cm:
Corvula macrops; Co: Cynoscion othonopterus, Cp: Cynoscion parvipinnis; Cpr: Cynoscion praedatorius; Cr: Cynoscion reticulatus; Gl : Genyonemus lineatus; Ir: Isopisthus
remifer; Ma: M. altipinnis (this study); Mec: M. ectenes (this study); Mm: M. megalops (this study); Mf: M. furnieri (Atlantic); Mun: M. undulatus (Atlantic); Mn:
Menticirrhus nasus; Me: Menticirrus elongatus; Mu: Menticirrus undulatus; Os: Ophioscion scierus; Ov: Ophioscion vermicularis; Rs: Roncador stearnsii; Sp: Seriphus politus;
Se: Stellifer ericymba; So: Stellifer oscitans; Tm: Totoaba macdonaldi; Ur: Umbrina roncador; Ub: Umbrina bussingi; Ux: Umbrina xanti. Dashed line indicates 2% of gen-
etic divergence as a cutoff criterion.

relationships with M. furnieri lineages. Shared lineages
between M. altipinnis and M. ectenes morphotypes (H2, H4, H6,
and H8) indicate that both are the same species. Singletons
between M. ectenes and M. megalops morphotypes suggest a
relatively recent evolution of lineages (H9, H11, and H12) from
the same population. This agrees with Freeland (2007) who
suggests that such disjunctions reflect high levels of gene
flow, where mutations spread before giving rise to new haplo-
types. Such lineages from M. ectenes originated in M. megalops
and those lineages, observed in M. altipinnisM. ectenes, sug-
gest gene flow over long distances. Despite a very low diver-
gence between central lineages of M. altipinnisM. ectenes and
M. megalops, a hint in structure between M. altipinnisM.
ectenes lineages (south of the TEP), which were not shared
with M. megalopsM. ectenes lineages (north of the TEP), sug-
gest population differences. Such population differences coin-
cide with the Cortez Province to the north of the TEP or the
Gulf of California and the Panamic Province south of the TEP, as
described by Robertson and Cramer (2009).
Lo et al. (2015) suggest that the earliest occurrence of
Sciaenidae is in tropical America, especially the Atlantic region.
Afterwards, this family invaded the eastern Pacific through the
Panama Seaway before the closure of the Isthmus of Panama
(3.1 Ma). In this scenario, the time between Micropogonias line-
ages from the TEP and M. furnieri from the Atlantic
(1.21.9 Ma) are in agreement with this vicariant event and are
of the same order of magnitude as the estimated time between
M. ectenes and M. furnieri (see Figures 3 and 4 in Lo et al. 2015).
Estimated divergence among M. altipinnisM. ectenes and
M. megalopsM. ectenes lineages (218336 ka) were similar
to those reported in other fish species (spotted bass
Paralabrax maculatofasciatus, orange throat pikeblenny
Chaenopsis alepidota, blue-banded goby Lythripnus dalli)
using cytochrome b and control region mtDNA fractions.
Such divergence times were obtained for disjunct populations
of the same species (Bernardi et al. 2003).
Considering the biogeographic evidence in the TEP, evolu-
tionary time estimates, and the presence of three lineages of
Micropogonias in sympatry, our results suggest the presence
of different populations of the same species with gene flow
over long distances, but under incipient speciation based on
environmental and ecological differences between provinces,
in the absence of isolation.
From the available information, it is our opinion that
Micropogonias is a single species in the Mexican coastal area
of the tropical Eastern Pacific (TEP), and that M. altipinnis
(Gunther 1864) should be the senior synonym for M. ectenes
(Jordan & Gilbert 1882) and M. megalops (Gilbert 1890).
Acknowledgements
Oswaldo Morales-Pacheco of Centro Regional de Investigacio n
Pesquera (CRIP) of Salina Cruz, Oaxaca who assisted with sampling of
M. altipinnis. Jose M. Grijalva-Chon of the Universidad de Sonora
donated five specimens of M. megalops. Ira Fogel of CIBNOR provided
editorial services. MEXBOL provided financial support to assist at the
Second Fish Barcode of Life World Conference. N.D.V., L.S.V., J.L.O.G.,
and J.D.A. are fellows of EDI-IPN. L.S.V., J.L.O.G., and J.D.A are fellows
of COFAA-IPN. G.S.P. was a recipient of a BEIFI-IPN and a CONACYT
fellowship (No. 208401).
Disclosure statement
The authors report no conflicts of interest.

Funding
Funding was provided by IPN-SIP grants 1721 and 20160514, and SEP-
CONACYT 2014-236864. Additional funding was received by L.S.V. and
Miguel F. Lavn-Peregrina of CICESE from the David and Lucille Packard
Foundation (2010-36137).
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