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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 number and type of fin rays, the number of gill rakers, and/or
various relative measurements of body parts. Occasionally,
The Sciaenidae fish family (commonly called drums or
otoliths and other specific bones are used for species discrim-
croakers) is composed of 24 genera (Chao 1995) in the trop-
ination (Teletchea 2009).
ical eastern Pacific (TEP), a region that stretches from the Gulf
Identification of Micropogonias species in the TEP (Chao
of California to northern Peru (Briggs 1974). In the TEP, two
1995), based on distinctive characteristics, is difficult because
biogeographic provinces have been defined, the Cortez
they are very similar. Original descriptions of the three
Province (Gulf of California and lower Pacific Baja California
Micropogonias species were based on a few individuals col-
Peninsula) and the Panamic Province (south of the TEP),
based on faunal complexes, regional endemics, and three lected at three locations; two specimens of M. altipinnis in
functional groups of shore fish species (reef, soft-bottom, and Panama and Guatemala, four of M. ectenes at Sinaloa, Mexico,
coastal pelagic). This reflects speciation within each province and four of M. megalops at Sonora, Mexico. These and other
(Robertson & Cramer 2009). Among the provinces, the genus difficulties explain why researchers have attempted to develop
Micropogonias is represented by three valid species: M. alti- new methods for identifying fish species without relying only
pinnis (Gunther 1864), M. ectenes (Jordan & Gilbert 1882), and on morphological features (Teletchea 2009).
M. megalops (Gilbert 1890). These three species share, to Identification based on genetics compares sequences of
some extent, their geographic range (Chao 1995). The slender genes, usually mtDNA loci that are known to vary between
croaker (M. ectenes) ranges from the southern Gulf of species. Mitochondrial genes have a high copy number per
California and the southwestern coast of the Baja California cell, typically lack recombination (promoting the loss or fix-
Peninsula to Oaxaca, Mexico. The tallfin croaker (M. altipinnis) ation of mtDNA haplotypes), reducing diversity within spe-
ranges throughout the Gulf of California and the tip of the cies, and enabling species identification (Dawnay et al. 2007).
Baja California Peninsula to Peru. The bigeye croaker (M. meg- Additional mtDNA features include patterns of maternal
alops) ranges throughout the Gulf of California to Oaxaca, inheritance and rapid rates of evolutionary change in com-
Mexico (Figure 1). parison to nuclear DNA, making it a suitable tool for genetic
In general, the identification of fish species is based on studies among taxa of several fish groups (Turan 2008). DNA
external morphological features, including body shape, colour barcoding has shown that the mitochondrial DNA cyto-
pattern, scale size and count, the relative position of fins, 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. with gill nets at the four locations, frozen, and transported on
2005; Kim et al. 2010). ice to IPNCICIMAR for processing. Some specimens were
Recent studies based on morphological data and sequen- deposited in the fish collection of IPNCICIMAR (http://colec-
ces of mtDNA have suggested a single taxonomic entity in cion.cicimar.ipn.mx).
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; Morphological analysis
Martnez-Guevara et al. 2015). MtDNA is also used for identi- Identification was based on keys (Chao 1995) and original
fying cryptic species (Gambusia; Lara et al. 2010; Albula gil- diagnoses (Gu nther 1864; Jordan & Gilbert 1882; Gilbert 1890;
berti and A. esuncula; Pfeiler et al. 2011), and Indian and Jordan & Evermann 18961900). The number of gill rakers,
western Pacific types of Lutjanus russellii into two species barbels, and rays of the dorsal fin were counted. The position
(Chu et al. 2013). of the longest spine in relation to the first two rays of the
If the TEP contains three species of Micropogonias with dorsal fin, when the dorsal fin was folded towards the body,
some sympatry, then morphological data and DNA sequences was taken into account. We made 18 morphometric measure-
should differentiate them, supporting the current taxonomic ments of each specimen (Figure 2), using a vernier caliper at
classification of Micropogonias species. This study compares
small measurements, and a measuring ruler or measuring
morphological differences (meristic, morphometry of body,
tape at bigger measurements. The measurements were made
and otoliths) with DNA sequences (CO1 and 16S fractions of
as follows: (1) Head length from the tip of the snout to the
mtDNA and 28S of nDNA) among M. altipinnis, M. ectenes,
beginning of the operculum, (2) Pectoral fin length from the
and M. megalops.
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
Materials and methods length from the beginning dorsal fin to the beginning of the
dorsal rays of caudal fin, (5) Interocular length measured as
Sampling the distance between eyes, (6) Caudal fin length from the tip
We collected 125 specimens of the three Micropogonias spe- of the fin to the base of rays, (7) Horizontal eye diameter
cies at four locations along the coast of the TEP from from the anterior to the posterior edge of eye, (8) Head
September 2012 through June 2013: 43 specimens of M. meg- height from the operculum isthmus to the dorsal edge of the
alops at San Felipe, Baja California (31 010 N, 114 490 W); 31 head, (9) Pelvic fin length from the tip of the fin to the base
specimens of M. ectenes at La Paz, Baja California Sur (24 090 N, of rays, (10) Preorbital length from the tip of snout to the
110 190 W) and six at Mazatlan, Sinaloa (23 120 N, 106 250 W); anterior edge of eye, (11) Maximum body height, (12) Anal
and 45 specimens of M. altipinnis at Salina Cruz, Oaxaca fin length from the beginning to the end of the fin, (13)
(16 100 N, 95 100 W) (Figure 1). Fish were collected by fishermen Caudal peduncle length from the end of the anal fin to the
MITOCHONDRIAL DNA PART A 3

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-
Figure 3. Left sagitta otolith of Micropogonias specimen and its parts: 1: sulcus;
criminant analysis of body measurements was performed to 2: ostium; 3: cauda; 4: crista. The position of the otolith in reference to spec-
assess patterns of variation and identify morphological char- imens body: D: dorsal; V: ventral; A: anterior; P: posterior.
acters that separate two or more groups or putative species.
Sagittal otoliths of every fish were removed, cleaned, and amplified with C1 (50 -ACCCGCTGAATTTAAGCAT-30 ) and C2 (50 -
photographed under a stereomicroscope with an attached TGAACTCTCTCTTCAAAGTTCTTTTC-30 ) (Le^ et al. 1993). DNA
camera. Pictures of the internal proximal side were taken and amplifications were performed in a PCR thermal cycler under
standardized scale was used as a reference. Measurements on the following conditions: 2 min at 94  C, 35 cycles of 1 min at
the otolith pictures were done relative to sulcus, using 94  C, 1 min at 50  C (CO1), 53  C (16S), or 42  C (28S), 1 min
Image-Pro Plus 6.0 software. The sulcus was divided into the at 72  C, and a final extension of 4 min at 72  C. PCR products
ostium and cauda, the division was at the crista (Figure 3). (20 lL, 100 ng lL1) were sequenced in sense and antisense
Six measurements obtained from the automated menu of the directions in an automatic sequencer (ABI Prism 3730XL,
software and 21 ratios of lengths and total area were com- Applied Biosystems, Carlsbad, CA) at Macrogen (Seoul, South
pared for the three species by discriminant analysis. Korea).
CO1 and 16S sequences of three Micropogonias species
from the GenBank were used as references (Table 1). The
Genetic analysis
best model of nucleotide substitution was selected with
We prepared 25 samples of muscle tissue: M. altipinnis MEGA 6.0 software (Tamura et al. 2013) for the construction
(n 10), M. ectenes (n 10), and M. megalops (n 5). of the maximum-likelihood tree. Genetic distances of K2P G
Genomic DNA extractions were carried out following a modi- (Kimura-two-parameter Gama) among reference sequences
fied protocol of Lopera-Barrero et al. (2008). Prior to PCR, of CO1 and 16S fractions and sequences of M. altipinnis, M.
DNA concentrations of samples were standardized to ectenes, and M. megalops were used to display phylogenetic
50100 ng lL1. The partial sequences of mtDNA genes: CO1 relationships by the maximum-likelihood tree (5000 boot-
(700 bp) and 16S rRNA (600 bp), and nDNA 28S (847 bp) were straps), using MEGA 6.0 software (Tamura et al. 2013).
amplified by PCR. The CO1 fraction was amplified with pri- Haplotype frequencies of CO1 were obtained for each
mers: VF1 (50 -TTCTCAACCAACCACAAAGACATTGG-30 ) (Ivanova Micropogonias species from the TEP and M. furnieri and M.
et al. 2007) and FishR1 (50 -TAGACTTCTGGGTGGCCAAAG undulatus from the Atlantic, using DnaSP 5.10 (Librado &
AATCA-30 ) (Ward et al. 2005). 16S rRNA (16S) was amplified Rozas 2009). A maximum parsimony haplotype network,
with 16Sa (50 -CGCCTGTTTAACAAAAACAT-30 ) and 16Sb (50 - using the Network 5 genetics software (Bandelt et al. 1999;
CCGGTTTGAACTCAGATCACGT-30 ) (Palumbi 1996). 28S was Polzin & Daneschmand 2003) was obtained with the CO1
4 
G. SANCHEZ-PINEDO ET AL.

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

Results
Genetic analysis
Morphological analysis
We obtained 25 sequences of CO1 (572 bp), 25 of 16S
The number of gill rakers in the first-gill arch ranged from (543 bp), and 25 of 28S (277 bp) for the three species (M.
1417 in M. megalops, 1216 in M. ectenes, 1315 in M. alti- megalops, M. ectenes, and M. altipinnis). All sequences were
pinnis. The ranges overlapped. The number of dorsal fin rays deposited in GenBank (Accession numbers: KX401587
showed a range from 2628 in M. megalops, 2427 in M. KX401611, KX417648KX417697).
ectenes, and 2123 in M. altipinnis. These ranges showed dif- The fraction of the mtDNA CO1 region of Micropogonias
ferences between M. megalops and M. altipinnis and between species from the TEP (10 individuals each), and Atlantic spe-
M. altipinnis and M. ectenes, but M. ectenes and M. megalops cies (five and four individuals of M. furnieri and M. undulatus,
overlap. Among the three species, all had four pairs of bar- respectively) revealed 23 haplotypes. Four haplotypes were
bels. The longest spine reaching the first two rays of the dor- shared between M. altipinnis and M. ectenes and four haplo-
sal fin when the dorsal fin was folded towards the body were types were unique to either species. Six haplotypes, including
none of the M. megalops, five of the 37 M. ectenes, and 38 of the most frequent, were unique to M. megalops. M. furnieri,
the 40 M. altipinnis. and M. undulatus, revealing three and two unique haplotypes,
Discriminant analysis with 17 morphometric variables respectively (Table 4).
(Table 2) showed three different morphological entities Intraspecific genetic divergences for CO1 ranged from
(F 34.212, Wilks c 0.002, p < .000). The dorsal fin length, 01.06% in M. megalops and 00.88% in M. ectenes and M. alti-
longest spine length of the dorsal fin, and the preanal length pinnis. In Atlantic species, divergences ranged from 00.17% in
were among the five most discriminant variables (Table 2). In M. undulatus and 00.53% in M. furnieri. Interspecific genetic
this analysis, M. altipinnis and M. megalops were different; divergences from the TEP were low. Divergences between M.
M. ectenes showed an intermediate morphotype, and ectenes and M. megalops ranged from 0.171.42%, between M.
M. altipinnis was the most distinctive species (Figure 4). altipinnis and M. ectenes from 01.24%, and between M. altipin-
Of 27 variables of otoliths, 11 had differences; 4 were nis and M. megalops from 0.351.78%. Comparisons among
automatically detected by the software and 7 were length Atlantic and TEP species showed divergences that ranged from
and total area ratios (Table 3). Discriminant analysis in groups 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 likelihood tree, using 28S, was not constructed because no
and 28S sequences, the highest interspecific divergence among variations were observed in this fraction.
the TEP and Atlantic species was 1.6% and 0%, respectively. The parsimony network (Figure 7) revealed that
The maximum-likelihood tree, using the CO1 sequence Micropogonias haplotypes from the TEP (H2, H6, H11, and
(Figure 6), groups M. ectenes, M. megalops, and M. altipinnis H13) had the fewest mutation steps (1719 mutations) com-
in the same clade, with a 69% of bootstrap. Atlantic species pared with haplotype 19 (H19) of M. furnieri, than any haplo-
were grouped in different clades with high bootstrap percen- types of M. undulatus (3536 mutations, figure not shown).
tages. Micropogonias furnieri was grouped with 99% boot- Four haplotypes (H2, H4, H6, and H8) were shared by M. alti-
strap and M. undulatus with 100% bootstrap. Within the clade pinnis and M. ectenes. Haplotypes were not shared by M.
of species from the TEP, three groups with low bootstrap per- megalops and M. ectenes; however, three haplotypes of M.
centages (less than 50%) were present. At the top was a ectenes (H9, H11, and H12) were connected to M. megalops
group with M. ectenes and M. altipinnis. In the middle was haplotypes by singletons and connected to shared haplo-
a group of M. ectenes and M. megalops. At the bottom was types of M. altipinnisM. ectenes (H2 and H6) by five mutation
a group with M. ectenes and M. altipinnis. While M. altipinnis steps (0.87% of genetic divergence). Central haplotypes of M.
and M. megalops were not together in any clade, M. ectenes altipinnisM. ectenes and M. megalops with higher frequencies
was present in three groups with the other two species (H2 and H13, respectively) were different only by three muta-
(Figure 6). tional steps (0.52% of genetic divergence).
The maximum-likelihood tree, using 16S, showed one Since some haplotypes were shared or were different by
clade containing all TEP species, including the sequence of one mutation step, we decided to group some haplotypes
M. furnieri (AY603006). Only the sequence of M. undulatus and named as follows: lineages M. altipinnisM. ectenes
was outside this clade (figure not shown). The maximum- (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 vertebrae or dorsal and anal fin rays (Seymour 1956; Orska
Province) and the later found in the northern TEP (Cortez 1957). Thus, based on this and on current genetic informa-
Province) (Figure 7, Table 6). tion, meristic characteristics cannot be the only approach to
Estimated divergence time among Micropogonias furnieri establish taxonomic status of a species.
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. Genetic analysis
ectenes lineages ranged from 218 kya to 336 kya (Table 6).
Avise (2000) and Hebert et al. (2003) suggest that interspe-
cific genetic variation is higher than 2% and intraspecific is
Discussion less than 1%. Our results contrast with these ranges, as
Micropogonias interspecific divergences in the TEP were <2%,
Morphological analysis
with some even lower than intraspecific ones (Table 5).
Until now, differentiation of species of Micropogonias used Nevertheless, a comparison of CO1 sequences of 22
meristics and the position of the longest spine of the dorsal Sciaenidae species in the TEP (Table 7) and Micropogonias
fin (Chao 1995). We found that meristics revealed latitudinal species in the TEP and Atlantic indicate that interspecific
variation in the gill rakers, rays of the dorsal fin, and the divergence ranges from 2.6819.29% and the range of intra-
number of fish with the longest spine reaching the first two specific divergence is 01.2% (Figure 8). These results suggest
rays of the dorsal fin. that Micropogonias species from the TEP conform to a single
Latitudinal variation in meristic characteristics, potentially species rather than three separate species.
associated with environmental and ocean-climate variations, The maximum-likelihood tree indicated significant differen-
have been documented in Micropogonias and other fish ces between Atlantic and TEP species, but no significant dif-
(Lindsey 1954; Vazzoler 1971; Figueroa & Daz de Astarloa ferences among Micropogonias in the TEP, supported these
1991; Silva-Segundo et al. 2011). In M. furnieri, differences in observations, notwithstanding that M. altipinnis and M. mega-
the number of gill rakers, lateral line scales, and/or fin rays lops did not share haplotypes. Silva-Segundo et al. (2011)
were reported between northern and southern populations report similar low genetic differences (0.40.8%) among three
of Brazil (Vazzoler 1971) and Uruguay (Figueroa & Daz de hake Merluccius species. They suggest that these should not
Astarloa 1991). These types of latitudinal variations have also be considered separate species because there was no mono-
been reported in the paradise gourami Macropodus opercula- phyletic clade, as defined by the phylogenetic species con-
ris (Lindsey 1954), and the Pacific hake Merluccius spp. cept (De Queiroz 2007). Rather, the tree reflects two
(Silva-Segundo et al. 2011). Earlier, controlled experiments in population units with some degree of gene flow. Similarly,
salmonids showed that the variation in environmental param- Byrkjedal et al. (2008) found non-significant differences for
eters (temperature, oxygen) could modify the number of 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 Paralabrax maculatofasciatus, orange throat pikeblenny
between M. altipinnis and M. ectenes morphotypes (H2, H4, H6, Chaenopsis alepidota, blue-banded goby Lythripnus dalli)
and H8) indicate that both are the same species. Singletons using cytochrome b and control region mtDNA fractions.
between M. ectenes and M. megalops morphotypes suggest a Such divergence times were obtained for disjunct populations
relatively recent evolution of lineages (H9, H11, and H12) from of the same species (Bernardi et al. 2003).
the same population. This agrees with Freeland (2007) who Considering the biogeographic evidence in the TEP, evolu-
suggests that such disjunctions reflect high levels of gene tionary time estimates, and the presence of three lineages of
flow, where mutations spread before giving rise to new haplo- Micropogonias in sympatry, our results suggest the presence
types. Such lineages from M. ectenes originated in M. megalops of different populations of the same species with gene flow
and those lineages, observed in M. altipinnisM. ectenes, sug- over long distances, but under incipient speciation based on
gest gene flow over long distances. Despite a very low diver- environmental and ecological differences between provinces,
gence between central lineages of M. altipinnisM. ectenes and in the absence of isolation.
M. megalops, a hint in structure between M. altipinnisM. From the available information, it is our opinion that
ectenes lineages (south of the TEP), which were not shared Micropogonias is a single species in the Mexican coastal area
with M. megalopsM. ectenes lineages (north of the TEP), sug- of the tropical Eastern Pacific (TEP), and that M. altipinnis
gest population differences. Such population differences coin- (Gunther 1864) should be the senior synonym for M. ectenes
cide with the Cortez Province to the north of the TEP or the (Jordan & Gilbert 1882) and M. megalops (Gilbert 1890).
Gulf of California and the Panamic Province south of the TEP, as
described by Robertson and Cramer (2009). Acknowledgements
Lo et al. (2015) suggest that the earliest occurrence of
Oswaldo Morales-Pacheco of Centro Regional de Investigacio n
Sciaenidae is in tropical America, especially the Atlantic region.
Pesquera (CRIP) of Salina Cruz, Oaxaca who assisted with sampling of
Afterwards, this family invaded the eastern Pacific through the M. altipinnis. Jose M. Grijalva-Chon of the Universidad de Sonora
Panama Seaway before the closure of the Isthmus of Panama donated five specimens of M. megalops. Ira Fogel of CIBNOR provided
(3.1 Ma). In this scenario, the time between Micropogonias line- editorial services. MEXBOL provided financial support to assist at the
ages from the TEP and M. furnieri from the Atlantic 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
(1.21.9 Ma) are in agreement with this vicariant event and are of COFAA-IPN. G.S.P. was a recipient of a BEIFI-IPN and a CONACYT
of the same order of magnitude as the estimated time between fellowship (No. 208401).
M. ectenes and M. furnieri (see Figures 3 and 4 in Lo et al. 2015).
Estimated divergence among M. altipinnisM. ectenes and
Disclosure statement
M. megalopsM. ectenes lineages (218336 ka) were similar
to those reported in other fish species (spotted bass The authors report no conflicts of interest.
MITOCHONDRIAL DNA PART A 11

Funding Lara A, Ponce de Leo n JL, Rodrguez R, Casane D, Co ^te G, Bernatchez L,


Garca-Machado E. 2010. DNA barcoding of Cuban freshwater fishes:
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