Population structure and identification of two matrilinear and one

Estuarine, Coastal and Shelf Science xxx (2014) 1e10
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Estuarine, Coastal and Shelf Science
journal homepage: www.elsevier.com/locate/ecss
Population structure and identification of two matrilinear and one
patrilinear mitochondrial lineages in the mussel Mytella charruana
Thainara Oliveira de Souza a, 1, Francisco Arimateia dos Santos Alves a, b, 1,
Colin Robert Beasley c, Luiz Ricardo Lopes de Simone d,
Nelane do Socorro Marques-Silva a, Guilherme da Cruz Santos-Neto a, e,
Claudia Helena Tagliaro a, *
, Campus de Bragança, Instituto de Estudos Costeiros, Laborato
~o e Biologia Evolutiva, Alameda Leandro
rio de Conservaça
Universidade Federal do Para
Ribeiro, s/n, Bragança, PA, CEP 68600-000, Brazil
b
~o de Meio Ambiente, Laborato
^ndia, Ananindeua, PA, CEP
rio de Biologia Ambiental, Rodovia BR 316, Km 07, s/n, Levila
Instituto Evandro Chagas, Seça
67030-070, Brazil
c
, Campus de Bragança, Instituto de Estudos Costeiros, Laborato
rio de Moluscos, Alameda Leandro Ribeiro, s/n, Bragança, PA,
Universidade Federal do Para
CEP 68600-000, Brazil
d
~o Paulo, Caixa Postal 42494, Sa
~o Paulo, SP, CEP 04299-970, Brazil
Museu de Zoologia da Universidade de Sa
e
, Campus de Abaetetuba, Rua Rio de Janeiro, 3322, Abaetetuba, PA, CEP 68440-000, Brazil
Instituto Federal de Ci^
encia e Tecnologia do Para
a
a r t i c l e i n f o
a b s t r a c t
Article history:
Accepted 10 November 2014
Available online xxx
The mitochondrial gene cytochrome c oxidase subunit I (COI) was sequenced from Mytella charruana
(N ¼ 243) at 10 Brazilian coastal localities to search for cryptic species, doubly uniparental inheritance
and investigate genetic population structure and demography. Three haplogroups were found: two
matrilinear (A and B) in males and females, and one patrilinear (C) found only in males. The p-distances
were 0.0624 (A and B), 0.2097 (A and C) and 0.2081 (B and C). Coalescence of M. charruana occurred
around 12.5 Mya, and the origins of the lineages were 3.4 and 4 Mya (matrilinear A and B) and 51.2 Mya
(patrilinear), which split before the separation of the genera Perna and Mytella. All individuals from the
northern coast of Brazil belonged to haplogroup A, whereas haplogroup B predominated among individuals from the eastern and northeastern coasts, with one exception, Goiana. Haplogroup C was found
in males from the northern to the eastern coast. GenBank sequences of M. charruana from Colombia,
Ecuador and four populations introduced to the USA joined Brazilian haplogroup B. Nuclear gene 18SITS1 sequences confirmed that all specimens belong to the same species. Four populations from the
northern coast of Brazil were homogenous with evidence of recent population expansion. All populations
from the northeastern and eastern coasts of Brazil were significantly structured (pairwise FST and
AMOVA). The heterogeneity among Brazilian populations requires that relocation for aquaculture be
preceded by genetic identification of the haplogroups. Differences in salinity and temperature may have
selected for distinct lineages of mussels and changing conditions in coasts and estuaries may allow only
resistant lineages of mussel to persist with the loss of others. In the light of global climate change, more
detailed data on temperature, pH, salinity and local currents could help explain the genetic structuring
observed among populations of Brazilian M. charruana.
© 2014 Elsevier Ltd. All rights reserved.
Keywords:
genetic diversity
geographic isolation
population genetics
DUI
Mytilidae
1. Introduction
Mytella charruana (d'Orbigny, 1842) (Mytilidae, Bivalvia), the
charru mussel, is native to Central and South America where it
* Corresponding author.
E-mail address: tagliaro@ufpa.br (C.H. Tagliaro).
1
Both authors contributed equally to this research.
ranges, along the Pacific coast, from Guayamas, Mexico to southern
Ecuador and the Galapagos Islands (Rios, 1994; Cardenas and
Aranda, 2000) and, along the Atlantic coast, from Colombia to
Argentina (Keen, 1971). Introíni et al. (2010) listed the diverse
synonyms attributed to M. charruana, including Mytella falcata
d'Orbigny, 1846, Modiola falcata Von Ihering, 1897, Mytilus strigatus
Von Ihering, 1900, Mytilus arciformis Dall, 1909, Mytilus mundahuensis Duarte, 1926 and Modiolus falcatus Morretes, 1949.
http://dx.doi.org/10.1016/j.ecss.2014.11.009
0272-7714/© 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: de Souza, T.O., et al., Population structure and identification of two matrilinear and one patrilinear
mitochondrial lineages in the mussel Mytella charruana, Estuarine, Coastal and Shelf Science (2014), http://dx.doi.org/10.1016/j.ecss.2014.11.009
2
T.O. de Souza et al. / Estuarine, Coastal and Shelf Science xxx (2014) 1e10
Laboratory experiments show that Mytella charruana (synonym
of Mytella falcata) planktonic larvae begin to settle on the substrate
and Carvalheira, 1972),
only after at least seven days (Paranagua
thus allowing a high level of dispersion. The latter is common in
species with a long planktonic larval stage and results in greater
gene flow and low levels of population genetic structure (Hoskin,
1997; Murray-Jones and Ayre, 1997; Collin, 2001), in contrast to
species with direct development (Collin, 2001). Environmental
factors, such as temperature, salinity, physical barriers, long
geographic distances, and oceanic current patterns, may, however,
limit gene flow in species with planktonic development (Launey
et al., 2002).
The charru mussel is an euryhaline species (Leonel and Silva,
1988; Gillis et al., 2009) that tolerates salinities varying between
2 and 40 (Yuan et al., 2010). These bivalves may be found in shallow
coastal lagoons and in muddy areas of bays and estuaries (Sibaja,
1988) in water temperatures of between 6 C and 31 C (Brodsky
et al., 2009). Along the eastern coast of the United States, the
charru mussel is considered an invasive species and has been found
in Florida since 1986, most likely being introduced via ballast water
or encrusted on ship hulls (Gillis et al., 2009).
Molecular studies carried out on marine and freshwater bivalves
show evidence for the presence of two lineages of mitochondrial
DNA (mtDNA), one maternal (mtDNA F) and another paternal
(mtDNA M) in origin due to doubly uniparental inheritance (DUI) of
mtDNA (Skibinski et al., 1994; Hoeh et al., 1996; Theologidis et al.,
2008). In this pattern of inheritance, females are typically homoplasmic for mtDNA F and transmit it to both sons and daughters,
whereas males are heteroplasmic, carrying both mitochondrial
genomes, whereas mtDNA F is predominant in somatic cells and M
in gonadal cells, but transmitting mtDNA M only to their sons
(Hoeh et al., 1996, 2002; Zouros, 2013). Although both lineages
evolve independently (Hoeh et al., 1996; Breton et al., 2007),
mtDNA M evolution is more rapid (Hoeh et al., 1997; Passamonti
and Ghiselli, 2009). DUI has been widely studied in bivalves and
has been recorded in the Mytilidae (Breton et al., 2007; Passamonti
and Ghiselli, 2009; Zouros, 2013). Alves et al. (2012) detected
mitochondrial heteroplasmy due to DUI in Mytella charruana from
the Brazilian coast, showing evidence for the presence of this type
of inheritance in yet another species of mytilid. According to the
latter authors, intraspecific divergence between mtDNA F and M in
M. charruana cytochrome c oxidase subunit I (COI) varied between
20.5 and 20.8%. However, the DUI pattern of inheritance was not
found in three species of the mussel Perna (Wood et al., 2007) and,
according to Gillis et al. (2009), DUI appeared to be absent in
M. guyanensis and M. charruana. The discrepancy between the results of the latter two studies regarding M. charruana is explained
by Alves et al. (2012) as a consequence of the choice of primers used
by Gillis et al. (2009), that were only able to amplify COI fragments
of matrilinear lineages.
An ancient polymorphism (L and M groups) with a 7.3% COI
sequence divergence was found between populations of the mussel
Brachidontes pharaonis in the Mediterranean Sea and in the Red Sea
between which, despite differences in haplotype frequencies, gene
flow appears to be extensive (Sirna-Terranova et al., 2006). However, the population from Salina di Marsala had private haplotypes
from L and M groups in high frequencies and the researchers hypothesize that these haplotypes are selectively advantageous in
waters with high temperature and salinity. A more widespread
analysis of the genus Brachidontes by the same authors, using COI
and 16S rDNA mitochondrial sequences, revealed three geographically distinct monophyletic clades: B. pharonis from the Mediterranean and Red Seas, B. variabilis from the Indian Ocean that also
has DUI, and B. variabilis from the western Pacific Ocean, which
have divergence values corresponding to interspecific values in
other bivalves and thus belong to three cryptic species (SirnaTerranova et al., 2007).
Oliveira et al. (2005), using allozymes, found intraspecific
structure among populations of Mytella charruana and
M. guyanensis from the Brazilian coast, and, although analyzes
indicated limited gene flow, the observed structure was not according to the isolation by geographic distance model. These authors were unable to give reasons for the retention of gene flow
due to the lack of detailed studies of larval dispersion and settlement. On the other hand, Gillis et al. (2009) analyzing molecular data (COI) of two invasive populations of M. charruana in
Florida (USA) and two native populations from South America
(Cartagena in Colombia and Guayaquil in Ecuador), found distinct
haplotypes among the South American populations. Zardi et al.
(2007), sequencing COI, found two genetic lineages (a western
and an eastern one) of the brown mussel Perna perna on the
South-East coast of South Africa that could be explained by the
pattern of ocean currents in that region. Populations of bivalves
from other families have also been studied from the Brazilian
coast using COI gene sequences. Arruda et al. (2009) found heterogeneity among four populations of Anomalocardia brasiliana,
whereby one population presented isolation by distance (Ilha
Canela, Bragança) and another isolation due to physical barriers
(Camurupim). Population studies carried out by Lazoski et al.
(2011) with allozymes and COI sequences, show intraspecific genetic structure in the oysters Crassostrea brasiliana and Crassostrea
rhizophorae, which, according to the authors, follows the isolation
by distance model.
There are only a few genetic studies of mussels of the genus
Mytella (Oliveira et al., 2005; Gillis et al., 2009; Alves et al., 2012).
The present study is the first based on molecular data (COI and 18SITS1) from a wide range of populations along the Brazilian coast
and the objective of this paper is to investigate the genetic diversity,
population structure, and demographic history of the native mussel
species Mytella charruana. Moreover, our study aims to identify
useful DNA markers for genetically characterizing the species
M. charruana. As three very distinct groups of COI sequences were
found, this led us to investigate the possibility of the presence of
cryptic species, an ancient polymorphism and/or doubly uniparental inheritance, all of which have previously been described in
Foighil, 2004; Sirne Terranova et al., 2006,
the Mytilidae (Lee and O
2007; Alves et al., 2012).
2. Material and methods
A total of 243 individual mussels were sampled at 10 localities
along the Brazilian coast, from Oiapoque on the northern coast, to
Antonina, on the eastern coast. The sample size ranged from 15 to
30 at each locality (Table 1). Whole specimens were conserved in
92% ethanol and frozen at 20 C pending laboratory analyzes. In
order to determine which COI haplotypes are patrilinear and which
are matrilinear, the sex of each individual was determined in six
specimens of Mytella charruana from Bragança and ten from Maceio
using hematoxilin-eosin dye and the methods described by Alves
et al. (2012). The sex of charru mussels from Antonina (N ¼ 10)
was determined by using fresh gonad smears by adding one drop of
distilled water to the gonad material, which was examined under
the light microscope (400). All mussels were morphologically
identified to species and the shells were deposited in the Laborio de Conservaça
~o e Biologia Evolutiva of the Universidade
rato
(LCBE/UFPA) or in the Museu de Zoologia da UniFederal do Para
~o Paulo (MZSP).
versidade de Sa
DNA was extracted from adductor muscles using the phenolchloroform protocol of Sambrook and Russell (2001). DNA from
gonads was extracted when the muscle sequences (mitochondrial
Please cite this article in press as: de Souza, T.O., et al., Population structure and identification of two matrilinear and one patrilinear
mitochondrial lineages in the mussel Mytella charruana, Estuarine, Coastal and Shelf Science (2014), http://dx.doi.org/10.1016/j.ecss.2014.11.009
T.O. de Souza et al. / Estuarine, Coastal and Shelf Science xxx (2014) 1e10
3
Table 1
Sampling locations, geographic coordinates, number of specimens in each haplogroup, number of haplotypes, and haplotype and nucleotide diversities for the ten populations
of Mytella charruana from the Brazilian coast, based on COI gene sequences.
Locations
Coordinates
Samples (N)
Specimens from
haplogroup A (NA)
Specimens from
haplogroup B (NB)
Number of distinct
haplotypes (NH)
Haplotype
diversity (h)
Nucleotide
diversity (p)
Oiapoque
~o Joa
~o de Pirabas (1)
Sa
Bragança (2)
^a (3)
Augusto Corre
Viseu (4)
Northern Coast (1 þ 2þ3 þ 4)
Fortim
Cabedelo
Goiana
Maceio
Antonina
04 020 0800 N51 120 000 W
00 460 08.8700 S47 100 47.0800 W
00 520 2600 S46 380 5900 W
01 010 12.1400 S46 390 12.6800 W
01 000 38.8200 S46 200 4.8600 W
e
02 510 25.3200 S40 050 28.500 W
06 580 4900 S34 490 4900 W
07 350 S34 500 W
09 380 00.2500 S35 460 00.1900 W
25 270 1700 S48 400 43.400 W
25
22
24
26
15
87
29
23
25
30
24
25
22
24
26
15
87
0
0
21
5
1
0
0
0
0
0
0
29
23
4
25
23
2
5
11
11
6
28
4
2
10
6
2
0.153
0.407
0.670
0.674
0.571
0.587
0.502
0.087
0.720
0.703
0.083
0.0003
0.0011
0.0032
0.0023
0.0016
0.0021
0.0024
0.0002
0.0190
0.0199
0.0052
COI) suggested heteroplasmy and from individuals of known sex.
Part of the mitochondrial COI gene was amplified using the polymerase chain reaction (PCR) with primers designed by Folmer et al.
(1994). COI sequences were compared with Mytella charruana sequences deposited in the GenBank (JQ685156 to JQ685159) by Alves
et al. (2012) to identify those belonging to the matrilinear or patrilinear lineages. To identify cryptic species, especially in families of
bivalves that are known to have DUI, a nuclear gene must be
sequenced in order to confirm if differences observed among the
mitochondrial sequences represent evolution of the species under
study or simply that of the mitochondrial genome. The nuclear DNA
fragment, the 18SrRNA (partial) and the internal transcribed spacer
1 (18S-ITS1), was chosen as the latter is already recognized as a
useful species specific marker in Mytilidae (Santaclara et al., 2006;
Wood et al., 2007; Alves et al., 2012). A subsample of 29 individuals
of both sexes from three COI haplogroups (NA ¼ 14; NB ¼ 6; NC ¼ 9)
was sequenced for 18S-ITS1 with primers designed by Pleyte et al.
(1992) and the protocols used for the amplification of the COI and
18S-ITS1 regions are described in Alves et al. (2012). Amplification
reaction products were sequenced for both strands on an ABI 3500
(Applied Biosystems) using the dideoxiterminal florescent marker
(BigDye® Terminator v3.1 Cycle Sequencing Kit) according to the
manufacturer's instructions.
DNA sequences were edited and aligned using Clustal W
(Thompson et al., 1997) in BioEdit 7.1.11 (Hall, 1999). The haplotype
network was obtained using COI sequences of mussels sampled in
the present study, in addition to sequences of Mytella charruana
deposited by Gillis et al. (2009) in GenBank (EU91742 to EU917180)
from Colombia, Ecuador and the USA. Haploviewer (Salzburger
et al., 2011) was used to construct the COI haplotype network in
order to to establish the relationship between different haplotypes
considering only variable sites. M and F lineages in COI sequences
were identified by direct comparison with COI sequences in GenBank (JQ685156, JQ685159) published by Alves et al. (2012), which
showed p-distances ranging from 20.5% to 20.8%. In our study, COI
sequences were found to belong to both the mtDNA F and mtDNA M
lineages and those belonging to the latter were removed from
population analyzes since these evolve independently (Hoeh et al.,
1996; Breton et al., 2007).
Nucleotide frequencies, termination codons and changes in
amino-acids were verified with Mega 5.0 (Tamura et al., 2011).
Identification of haplotypes, grouping of individuals by population,
and the generation of the file to be run in Arlequin 3.5.1.2 (Excoffier
and Lischer, 2010) were carried out using DNASp 4.1 (Rozas et al.,
2003). Arlequin 3.5.1.2 (Excoffier and Lischer, 2010) was further
used for the following analyzes. The indexes of haplotype and
nucleotide diversities were calculated, and the former is defined as
the probability that two randomly chosen haplotypes are different
in the sample and the latter as the probability that two randomly
chosen homologous nucleotide sites are different (Nei, 1987). Tajima's (D, Tajima, 1989) and Fu's (FS, Fu, 1997) tests were used to
check if the population was in a state of selective neutrality.
Negative and significant values of D (Tajima's test) are suggestive of
purifying selection, recent increase in population size, and population subdivision (Tajima, 1993). Negative and significant values of
FS (Fu's test) indicate recent population expansion or hitchhiking
(Fu, 1997). The hypothesis of mussel population expansion in the
present study was investigated by analysis of the distribution of
pairwise differences in the population (mismatch distribution). A
unimodal mismatch distribution is evidence that the population is
going through a rapid and sudden expansion (Rogers and
Harpending, 1992). The significance of the differences between
these values was calculated from the sum of squared deviations
(SSD, Excoffier and Schneider, 1999) with 10,000 permutations. The
estimates of the time of expansion were made using the equation
t ¼ 2ut (Rogers and Harpending, 1992) and calibrated with a
divergence rate of 0.52% per nucleotide per million years estimated
for COI by Luttikhuizen et al. (2003) who used bivalve fossil record
dates. The generation time was estimated at 1 year (Pereira et al.,
2003).
Genetic structure among populations was tested by pairwise FST
and significance was checked using 10,000 permutations. The
partitioning of genetic variability was tested using analysis of molecular variance (AMOVA, Excoffier and Lischer, 2010) with 10,000
permutations. Both previous analyzes were developed in Arlequin
3.5.1.2 (Excoffier and Lischer, 2010). AMOVA was carried out
considering all populations together; and, due to the clear separation, by at least 30 mutations, of the matrilinear lineage in two
groups (haplogroups A and B) from Brazilian populations, by
pooling the populations in their haplogroups. Associations among
FST values and coastal geographic distances among Mytella charruana populations were tested considering all populations
together, and, separated by haplogroups. For this purpose, the
Mantel test (10,000 permutations) was carried out using IBDWS
online (Jensen et al., 2005). Geographic distances between populations were estimated using Google Earth by measuring the
distance along the coastline.
The evolutionary model and the parameters used to obtain the
maximum likelihood (ML) tree were selected by JModeltest
(Guindon and Gascuel, 2003; Posada, 2008). The significance of the
ML tree arrangements were tested by bootstrapping with 1000
pseudo-replicates using PhyML 3.0 (Guindon et al., 2010). Bootstrap
values equal or superior to 95% were considered statistically significant (Li, 1997). COI sequences from other Mytilidae were added
to the phylogenetic analysis (Genbank numbers: JQ685160,
JQ685162, GU570464, AY497292, JX486124).
Please cite this article in press as: de Souza, T.O., et al., Population structure and identification of two matrilinear and one patrilinear
mitochondrial lineages in the mussel Mytella charruana, Estuarine, Coastal and Shelf Science (2014), http://dx.doi.org/10.1016/j.ecss.2014.11.009
4
T.O. de Souza et al. / Estuarine, Coastal and Shelf Science xxx (2014) 1e10
A phylogenetic tree based on Bayesian Inference (BI), and using
COI sequences, was created in BEAST 2 (Bouckaert et al., 2014) to
estimate the time to the most recent common ancestor (tMRCA)
among mtDNA lineages of Mytella charruana and other Mytilidae.
The input file for BEAST 2 was created using the application
BEAUti. The phylogenetic tree used the strict clock model and the
prior was set to the default option of the Yule process (Drummond
et al., 2006). Fossil dating (Barsotti and Meluzzi, 1968; Coan et al.,
2000) of the separation of Mytilus galloprovincialis and Mytilus
edulis (2 Mya); and Mytilus californianus and M. edulis - M. galloprovincialis (30 Mya) were used as references to calculate the
tMRCA.
3. Results
In subsamples of Mytella charruana (N ¼ 26) there were four
females and two males in Bragança; eight females, one male and
; and one male, seven females
one of indeterminate sex in Maceio
and two individuals of indeterminate sex in Antonina. All specimens sequenced in this study were morphologically identified as
M. charruana, and the shells from all localities were deposited as
lots 109537 to 109545 and 109546 to 109551 in MZUSP and in
LCBE/UFPA. Sequences of the nuclear fragment 18S-ITS1 agree with
this identification when compared with the sequence JQ734971
from GenBank (Alves et al., 2012), with reference to lot number
99629 at MZUSP. The 18S-ITS1 showed four very similar sequences
with only one mutation and an indel of 1e2 nucleotides and were
not related to any COI haplogroups.
We unambiguously sequenced 510 bp of the COI gene (mtDNA F
lineage) from a total of 243 Mytella charruana and the COI sequences could clearly be divided into three very distinct haplogroups: A, B and C (Fig. 1). Comparison with sequences deposited
in GenBank (JQ685158, JQ685159) by Alves et al. (2012), made
possible the unambiguous identification of a patrilinear lineage
(haplogroup C) in the COI sequences of specimens collected in
Oiapoque (N ¼ 12), Bragança (N ¼ 4), Viseu (N ¼ 4), Cabedelo
(N ¼ 2) and Antonina (N ¼ 1). The
(N ¼ 2), Goiana (N ¼ 1), Maceio
comparison of COI sequences from mtDNA M (patrilinear) and
mtDNA F (matrilinear) lineages presented between 103 and 107
mutated sites (Fig. 1) and p-distances of 0.2081 and 0.2097,
respectively. The same patrilinear lineage (haplogroup C) was
found in males with both matrilinear lineages (haplogroup A and
B). Haplogroup C had eight distinct haplotypes (H47 to H54) with
two to seven nucleotides and one to two amino acid mutations
between them.
COI sequences were obtained from a total of 243 Mytella charruana, varying from 15 to 30 individuals at each of the 10 localities
(Table 1). Among all populations, 46 haplotypes were identified
from the mtDNA F lineage (GenBank accession numbers KP013759
to KP013804), which were classified, according to the network division, into two distinct haplogroups (Fig. 1). As seen in Fig. 2,
haplogroup A was predominant in mangroves from northern Brazil,
from Oiapoque to Viseu, whereas haplogroup B was predominant
from Fortim (northeast) to Antonina (east), with the exception of
Goiana (northeast) where sequences from haplogroup A predominated (N ¼ 21) over those from B (N ¼ 4). Haplogroup A was
separated from haplogroup B by at least 30 mutations (p-distance:
0.0624). In haplogroup A, 38 haplotypes were found, of which H1
was the most frequent (N ¼ 79), followed by H30 (N ¼ 19). In
haplogroup B, a total of 8 haplotypes was found, of which H39
(N ¼ 60) was the most common, followed by H44 (N ¼ 23) and H41
(N ¼ 12). The remaining haplotypes of both haplogroups were
found in sequences from 1 to 3 individuals. Within haplogroups,
the number of mutations varied from 1 to 7 for group A and from 1
to 4 for B (Fig. 1). Gillis et al. (2009) COI sequences of M. charruana
from GenBank (EU917142 to EU917180) were related to haplogroup
B (Figs. 1 and 2), which predominated in eastern Brazilian populations, but, however, did not share haplotypes with any of the
Brazilian populations. After inclusion of the haplotypes from Gillis
et al. (2009) study, haplogroups A and B were separated by 26
mutations. Therefore, based on the network arrangements, the
M. charruana COI sequences were divided into two matrilinear
haplogroups (A and B) and one patrilinear (C). In haplogroup A, the
haplotype distribution from northern Brazil and from Goiana
exhibit a star shaped network suggestive of recent demographic
expansion (Grant and Bowen, 1998).
Fig. 1. The haplotype network divided the COI sequences (510 bp) from 10 populations of Mytella charruana from the Brazilian coast into two matrilinear and one patrilinear groups,
and showed that the matrilinear group B is also present in sequences from Colombia, Ecuador and the United States of America (USA) obtained from Gillis et al. (2009). H1 to H46:
Brazilian matrilinear haplotypes. Letters: Haplotypes from Colombia, Ecuador and USA.
Please cite this article in press as: de Souza, T.O., et al., Population structure and identification of two matrilinear and one patrilinear
mitochondrial lineages in the mussel Mytella charruana, Estuarine, Coastal and Shelf Science (2014), http://dx.doi.org/10.1016/j.ecss.2014.11.009
T.O. de Souza et al. / Estuarine, Coastal and Shelf Science xxx (2014) 1e10
5
Fig. 2. Sampling locations of Mytella charruana and proportions of haplogroups A and B at each location.
In the Antonina population (east coast), 23 individuals had
haplotype H44, found only at this locality and belonging to haplogroup B, and one specimen had haplotype H30, from haplogroup
A. Individuals from the east coast of Brazil from haplogroup A
, N ¼ 5; Antonina, N ¼ 1) do not share the
(Goiana, N ¼ 21; Maceio
same haplotypes with individuals from the north coast, there being
at least two mutations between them.
The global analysis showed intrapopulation haplotype and
nucleotide diversities ranging from 0.083 (Antonina) to 0.720
), respectively
(Goiana), and 0.0002 (Cabedelo) to 0.0199 (Maceio
and Antonina,
(Table 1). Only three populations, Goiana, Maceio
showed haplotypes from both haplogroups (A and B). A second
analysis, considering only haplotypes belonging to the predominant haplogroup was carried out for these populations and, in this
case, the haplotype diversities ranged from 0.000 (Antonina) to
0.629 (Goiana) and the nucleotide diversities from 0.0000 (Anto ). The nucleotide diversities were low
nina) to 0.0030 (Maceio
among haplotypes of the same haplogroup but high in comparison
to those of other haplogroups.
Genetic structure (FST) was found among the majority of pairwise populations using all haplotypes present in the populations
and using only haplotypes of the predominant haplogroup in each
population (Table 2). AMOVA among all populations and considering all haplotypes corroborated these results showing that 82.07%
of the variation was among populations (FST ¼ 0.820; P < 0.001).
~o Joa
~o
However, populations from the northern coast, between Sa
de Pirabas and Viseu, were homogenous (FST ¼ 0.005; P ¼ 0.227),
but when Oiapoque is added to this group, AMOVA indicates heterogeneity (FST ¼ 0.013; P ¼ 0.050). AMOVA between haplogroups
A and B showed heterogeneity, responsible for 85.84% of the variation and also among the populations within both groups F
(FCT ¼ 0.858; P ¼ 0.004).
When genetic (FST values) and geographic distances of all populations of Mytella charruana were compared, Mantel test results
Table 2
Pairwise FST value comparisons among populations of Brazilian Mytella charruana. Lower diagonal: the analysis was carried with all individuals from each population. Upper
diagonal: only individuals from the predominant haplogroup were used in each population.
Locations
Oiapoque
~o de Pirabas
S~
ao Joa
Bragança
^a
Augusto Corre
Viseu
Goiana
Fortim
Cabedelo
Maceio
Antonina
Oiapoque
~o Joa
~o de Pirabas
Sa
Bragança
^a
Augusto Corre
Viseu
Goiana
Fortim
Cabedelo
Maceio
Antonina
e
0.02560
0.03732*
0.02975
0.03017
0.29046*
0.97573*
0.99595*
0.77984*
0.95212*
0.02560
e
0.01445
0.00453
0.01109
0.26573*
0.96910*
0.98979*
0.76357*
0.94305*
0.03732*
0.01445
e
0.00227
0.01408
0.24813*
0.95454*
0.97193*
0.75755*
0.92793*
0.02975
0.00453
0.00227
e
0.00720
0.27061*
0.95997*
0.97770*
0.76626*
0.93478*
0.03017
0.01109
0.01408
0.00720
e
0.22305*
0.96405*
0.98787*
0.73323*
0.93282*
0.81267*
0.74656*
0.59924*
0.66175*
0.70890*
e
0.81009*
0.81442*
0.57721*
0.76632*
0.97573*
0.96910*
0.95454*
0.95997*
0.96405*
0.96717*
e
0.13016*
0.12852*
0.44002*
0.99595*
0.98979*
0.97193*
0.97770*
0.98787*
0.98626*
0.13016*
e
0.16801*
0.57617*
0.97155*
0.96401*
0.94834*
0.95445*
0.95752*
0.96194*
0.05349
0.31709*
e
0.19592*
0.99734*
0.99129*
0.97335*
0.97907*
0.98965*
0.98769*
0.69460*
0.97872*
0.66536*
e
*P < 0.05.
Please cite this article in press as: de Souza, T.O., et al., Population structure and identification of two matrilinear and one patrilinear
mitochondrial lineages in the mussel Mytella charruana, Estuarine, Coastal and Shelf Science (2014), http://dx.doi.org/10.1016/j.ecss.2014.11.009
6
T.O. de Souza et al. / Estuarine, Coastal and Shelf Science xxx (2014) 1e10
Table 3
Populations of Mytella charruana from the Brazilian coast classified by haplogroup with details of sample size, number of haplotypes, and results of Tajima's (D) and Fu's (Fs)
tests, sum of squared deviations (SSD) and tau (t).
t
Locations
N
Haplotypes (N)
D (P)
FS (P)
SSD (P)
Oiapoque
~o Jo~
Sa
ao de Pirabas (1)
Bragança (2)
^a (3)
Augusto Corre
Viseu (4)
Homogenous Northern Coast populations (1þ2þ3þ4)
Fortim
Cabedelo
Goiana
Maceio
Antonina
Only haplotypes from Haplogroup A
Goiana
Only haplotypes from Haplogroup B
Maceio
Antonina
25
22
24
26
15
87
29
23
25
30
24
2
5
11
11
6
28
4
2
10
6
2
0.69827 (0.22780)
1.80901 (0.01510)
2.00002 (0.00840)
2.08909 (0.00550)
1.98306 (0.00820)
2.55797 (0.00000)
0.11134 (0.49060)
1.16097 (0.14650)
0.24363 (0.45270)
0.65598 (0.79310)
2.60844 (0.00010)
0.28345 (0.17260)
2.66224 (0.00740)
6.46281 (0.00010)
8.04625 (0.00000)
3.53713 (0.00080)
29.68505 (0.00000)
0.88475 (0.71000)
0.99304 (0.07140)
2.45687 (0.85030)
9.31976 (0.99400)
7.17462 (0.99440)
0.02833
0.00066
0.01395
0.00036
0.00035
0.00045
0.35448
0.00003
0.05624
0.10793
0.01026
(0.10630)
(0.81150)
(0.76950)
(0.95910)
(0.82490)
(0.95830)
(0.95510)
(0.81460)
(0.41090)
(0.11640)
(0.84350)
2.965
0.500
2.961
1.447
0.848
1.916
0
3.000
1.078
31.586
3.000
21
9
1.84558 (0.01850)
7.53331 (0.00000)
0.00707 (0.42600)
0.955
25
23
5
1
1.20908 (0.89000)
0.00000 (1.00000)
0.28718 (0.59260)
0.00000 (N.A)
0.10957 (0.14830)
0.00000 (0.00000)
3.629
0
were significant (r ¼ 0.533, P ¼ 0.005). When separated by haplogroup, results were significant only for haplogroup A (r ¼ 0.905,
P ¼ 0.002), in contrast to haplogroup B (r ¼ 0.800, P ¼ 0.078).
Tajima's (D) and Fu's (FS) values (Table 3) revealed deviation
from neutrality with significant negative values in four populations
~o Joa
~o de Pirabas, Bragança, Augusto Corre
^a
from northern Brazil (Sa
and Viseu). As the latter four populations were homogenous, these
were grouped and both neutrality tests were recalculated, again
resulting in significant negative values. Antonina, located on the
east coast, deviated only for Tajima's test, probably due to a
bottleneck. The remaining populations did not present deviation
from selective neutrality. In three populations, individuals from
both haplogroups were found (Table 3). In Goiana, specimens from
and Antohaplogroup A predominated (84%) whereas in Maceio
nina, haplogroup B was the most frequent (83.3% and 95.8%,
respectively). A large difference in the frequency of one haplogroup
with respect to another in a population suggests recent mixture of
stocks, therefore Fu's and Tajima's tests were also carried out
without the haplotypes of the minority haplogroup for Goiana,
and Antonina. The latter analysis showed deviation of
Maceio
neutrality and signs of demographic expansion only for Goiana
(Table 3).
The northern coast group, comprising the four homogenous
populations, and Goiana (only haplogroup A) both showed unimodal pairwise mismatch distributions, with curves indicating recent
expansion (Fig. 3a and b, respectively). The sum of squared deviations (SSD) analysis (Table 3) supported the hypothesis of recent
demographic expansion in the northern coast group and in the
Goiana population. Considering a mutation rate of 0.52%
(Luttikhuizen et al., 2003), the northern coast group showed signs
of demographic expansion, which began around 360,000 years ago,
and Goiana, in the northeast, presented demographic expansion
dating from 180,000 years ago.
The Bayesian coalescent phylogenetic tree (Fig. 4) shows that
the coalescent time calculated for Mytella charruana was 12.5 Mya;
for haplogroup A 3.4 Mya and for haplogroup B 4.0 Mya; for
northern populations 2.8 Mya, and, for the Goiana population
1.9 Mya. The patrilinear lineage of M. charruana coalesced prior to
the origin of Perna perna (51.2 Mya).
The evolutionary model selected by JModeltest (Guindon and
Gascuel, 2003; Posada, 2008) for the Mytella charruana haplotypes was the K81 (Kimura, 1981) method and the proportion of
invariable sites was 0.7040. A Maximum Likelihood (ML) analysis
was ran and resulted in a phylogenetic tree with topology similar to
thatof the BI tree (Fig. 4), thus we included the bootstrap values of
ML in the BI tree. The arrangements of ML and BI trees supported
the results shown in the haplotype network (Fig. 1), dividing the
COI sequences into three haplogroups supported by bootstrap
values ranging from 92% to 100%. The relationships among matrilinear M. charruana, M. guyanensis and Perna perna were not supported by strong bootstrap values, however the basal position of
the patrilinear lineage is clear (bootstrap: 100%).
4. Discussion
COI sequences from Mytella charruana revealed two matrilinear
lineages and one patrilinear lineage. Mitochondrial sequence heteroplasmy was found in populations of both haplogroups of Mytella
Fig. 3. Observed distribution of pairwise differences, in comparison to the expected population growth model, based on COI gene sequences from Brazilian Mytella charruana. a)
four homogenous populations from the northern coast; b) population from Goiana (northeastern coast), including only haplotypes belonging to haplogroup A.
Please cite this article in press as: de Souza, T.O., et al., Population structure and identification of two matrilinear and one patrilinear
mitochondrial lineages in the mussel Mytella charruana, Estuarine, Coastal and Shelf Science (2014), http://dx.doi.org/10.1016/j.ecss.2014.11.009
T.O. de Souza et al. / Estuarine, Coastal and Shelf Science xxx (2014) 1e10
7
Fig. 4. Bayesian coalescent phylogenetic tree with the estimate of the time to the most recent common ancestor assuming a strict molecular clock, based on COI sequences for
haplogroups A, B and C of M. charruana and sequences of other Mytilidae with dates of the separation in million of years ago and confidence interval in parentheses. A previous
Maximum Likelihood analysis resulted in a phylogenetic tree with similar topology and bootstrap values (1000 pseudo-replicates) are shown preceding an asterisk.
charruana and comparisons with Alves et al. (2012) sequences
indicate the presence of DUI. As the patrilinear lineage of the
mtDNA evolves faster than the matrilinear (Passamonti and
Ghiselli, 2009; Zouros, 2013), only the latter is used in population
analyzes (Hurlwood et al., 2005; Arruda et al., 2009; Gillis et al.,
2009; Lazoski et al., 2011).
found in the 18S-ITS1 sequences may be related to concerted
evolution of repetitive genes with consequent homogenization of
this fragment (Elder-Jr and Turner, 1995). The absence of a second
patrilinear mitocondrial lineage may be due a lack of specificity of
the pair of primers used, perhaps because of a mutation peculiar to
this lineage.
4.1. Two matrilinear haplogroups: one species or two cryptic
species?
4.2. Geographic distribution of the haplogroups
The large differences among COI mitochondrial sequences from
the matrilinear lineages from Brazil resulted in haplotypes being
classified into two distinct haplogroups. Most populations had
moderate haplotype diversity and low nucleotide diversity, except
, in which haplotypes from the two distinct
Goiana and Maceio
haplogroups A and B are established, and in this case both haplotype and nucleotide diversities are high (Table 1). Although there
was no evidence of morphological differences among the Mytella
charruana sampled from the 10 localities along the Brazilian coast,
the presence of two mitochondrial haplogroups could suggest the
existence of two cryptic species. However, nuclear sequences of the
18S-ITS1 fragment, considered a useful marker for mytilid species
(Santaclara et al., 2006; Wood et al., 2007), did not reveal significant differences among the 18S-ITS1 sequences of specimens of
both matrilinear (A and B) and patrilinear (C) COI haplogroups, thus
supporting a single species hypothesis for M. charruana. Moreover,
the patrilinear sequences revealed only one haplogroup, C, present
in all populations also indicating that northern and northeasterneastern populations belong to the same species.
Even if a speciation process has begun in the charru mussel,
either by geographic isolation or ecological adaptation, the present
data suggest that individuals of different COI haplogroups are still
able to mix, since they share alleles from the 18S-ITS1 fragment.
The low variability, even after an initial diversification process,
The differences between the two matrilinear haplogroups of
Mytella charruana from the Brazilian coast also appear to be related
to geographic distribution, with the unique presence of one group
on the northern coast (haplogroup A) and the other predominant
along the northeastern and eastern coasts (haplogroup B). In populations of Perna perna from eastern and western South Africa, a
lesser degree of divergence, involving approximately 12 mutations,
was detected using COI sequences (400 bp) and a central zone,
where overlap occurred, was evident (Zardi et al., 2007). According
to the authors, maritime currents or local selective forces would
explain these differences. Similar results were found in comparisons of sequences of specimens of P. perna from South Africa with
those of Morocco and Mauritania, from North Africa (Wood et al.,
2007). Another species with two COI haplotype lineages is the Pacific oyster or Japanese oyster Crassostrea gigas, which, when
brought to Europe probably by accident, became known as Crassostrea angasi (Hurwood et al., 2005). However, despite the differences, the number of mutations in COI sequences (547 bp) between
both stocks, also around 12, does not justify their classification as
two distinct species (Reece et al., 2008).
The samples sequenced from Colombia by Gillis et al. (2009) are
related to haplogroup B, found in northeastern and eastern Brazil,
and not to populations from northern Brazil that are, in fact,
geographically closer, suggesting overlap of the distribution of the
haplogroups. However, in the present study, five locations were
Please cite this article in press as: de Souza, T.O., et al., Population structure and identification of two matrilinear and one patrilinear
mitochondrial lineages in the mussel Mytella charruana, Estuarine, Coastal and Shelf Science (2014), http://dx.doi.org/10.1016/j.ecss.2014.11.009
8
T.O. de Souza et al. / Estuarine, Coastal and Shelf Science xxx (2014) 1e10
sampled around the mouth of the Amazon river, between Oiapoque
and Viseu, and no evidence of haplotypes from haplogroup B was
found in this region. Comparisons among sequences from the
maternal lineage with those of Gillis et al. (2009), showed similarities between the Brazilian populations with haplogroup B,
predominant along the northeastern and eastern coasts, and four
invasive populations in the United States and two native ones, from
Cartagena (Colombia) and Guayaquil (Ecuador). However, none of
the Brazilian COI sequences were identical to those of Mytella
charruana in Gillis et al. (2009) suggesting that Brazilian stocks
were not involved in the invasions studied by the latter authors.
The specimens collected from Goiana (northeastern coast) were
from haplogroup A. However, this population was significantly
different from the remaining populations in this group, located on
the northern coast, showing that this is a distinct population,
probably due to a founder effect and isolation by distance. The direction of the maritime currents are from the northeast to the
North, via the North Brazil Current (Cirano et al., 2006), however,
only one individual from the northern coast (Bragança), presented a
haplotype (H16) derived from the Goiana stock (Fig. 1). This finding
suggests that the charru mussel from haplogroup B (predominant
in the Northeast and East) may be unable to survive in the mangroves of the North, which receive high freshwater discharges
during the rainy season. Salinity, in 2003, ranged from 10.9 to 40 in
the Bragança region, for example (Santos-Filho et al., 2008). On the
other hand, the population of the mussel Brachidontes pharaonis
from Salina di Marsala (Mediterranean Sea, Italy) had private
haplotypes in high frequencies that may be selectively advantageous in waters with high temperature and salinity (SirnaTerranova et al., 2006).
4.3. Demographic history
All northern populations from haplogroup A, except those from
Oiapoque, were homogenous and showed evidence of population
expansion dating from around 360,000 years ago. Moreover, the
Goiana Mytella charruana stock, which also belongs to haplogroup
A, showed significant population growth around 180,000 years
ago. Over the past 700,000 years, Earth has been subject to
widespread and long-lasting glaciations interrupted by shorter,
warmer interglacial periods that have lead to successive changes
in sea level, altering the shape of the coastline and the distribution
of coastal species (Hewitt, 1996). Consequently, many populations
may have suffered bottlenecks, been permanently isolated or
accumulated genetic differences by drift or selection for local
adaptations. Currently, rising CO2 concentrations are changing the
temperature and the acidity of the ocean and, along with changes
in salinity with variable freshwater inflow, relative sea level rise is
redesigning the coast line in many parts of the world (Roessig
et al., 2004).
The populations from the northeastern and eastern coasts,
where haplogroup B predominated, are in selective neutrality,
except for Antonina, where there was evidence of a bottleneck or
founder effect. In this locality, the exact sampling location, in the
mangrove, was relatively protected from the influence of oceanic
currents that may limit natural dispersion in the region. Cho et al.
(2007) found similar results with six populations of Scapharca
broghtonii from Korea that presented genetic differences among
which a single population (Jinhae) was differentiated in relation to
the others since it had limited dispersal with the presence of a
narrow avenue for exchange with marine currents. On the other
hand, the sampling location in Antonina was close to a busy port
and the presence of a single haplotype from haplogroup B (N ¼ 23)
and another from haplogroup A (N ¼ 1) may be explained by their
recent introduction through shipping. The transport of incrusting
bivalves on hulls and in ballast water is well known and has been
documented by Farrapeira et al. (2011) for the Brazilian coast.
With the current data, it was not possible to establish with
certainty the cause of the separation of Mytella charruana into two
mitochondrial haplogroups. The coalescence of Atlantic charru
mussels goes back a long time, around 12.5 Mya (CI: 8.3 to
17.3 Mya); and, 3.4 Mya (CI: 2 to 5 Mya) and 4 Mya (CI: 2.7 to
5.7 Mya) for haplogroups A and B, respectively. This was a period of
important changes when excessive global cooling severely reduced
the sea level and when, around 10.5 Mya, the direction of the
Amazon river was inverted (Figueiredo et al., 2009), which may
have drastically affected the ecosystem around its new mouth.
Although the changes in amino acids produced by the nucleotide
mutations in the COI gene were sporadic and did not represent
important differences between the haplogroups, the A lineage
mainly found around the mouth of the Amazon river, may represent adaptations to low salinity, high turbidity and other factors
characteristic of this coastal area.
4.4. Doubly-uniparental inheritance
Considering the existence of DUI in this species (Alves et al.,
2012), the hypothesis of role-reversals (Hoeh et al., 1997) may
arise. Could the presence of two matrilinear lineages be explained
by an unusual feminization of a mtDNA M? Could the absence of a
second patrilinear lineage be due to a recent masculinization of the
mtDNA F? Recent studies based on sequences of the control region
(CR) of Mytilus species showed large differences among matrilinear
and patrilinear mitochondrial sequences, the results indicated that
role-reversal is a rare event involving recombination of both genomes (Zouros, 2013). Moreover, feminization of the male genome
has never been described in bivalves, the matrilinear mtDNA
genome is under stronger selection than the patrilinear one, which
allows more frequent amino acid mutations that may eventually be
fixed in the latter lineage. It provides an explanation as to why
recent mtDNA masculinized genomes (F to M) have a higher chance
of success than feminized ones (M to F) (Hoeh et al., 1997; Zouros,
2013). Comparison of our data with those of Alves et al. (2012),
shows that, besides the high nucleotide mutation rates observed for
the COI gene of patrilinear origin, they also found a high rate of
change in amino acids, which was not found in lineages of both
matrilinear haplogroups in our study. Therefore, the data do not
support the occurrence of role-reversal from mtDNA.
4.5. Independent mitochondrial evolution or interrupted
speciation?
With the peculiar pattern of mitochondrial inheritance, where
recombination does not occur and the mitochondria are sent
directly from the mother to her offspring, lineages may evolve
independently until they disappear due to drift or natural selection
(Avise, 2000). However, if the population is numerous and the
mutations result in adaptive effects that do not lead to disadvantages, the population may retain more than one mitochondrial
lineage evolving independently for many generations. This last
hypothesis is probably the cause of the observed divergence.
4.6. Conclusions and advice for management
The present study, based on a substantial number of populations
and specimens, shows that mtDNA genes can be important tools in
evolutionary studies, but also that they should be used with caution
due to their particular pattern of matrilinear and non recombinant
inheritance, in addition to doubly-uniparental inheritance in some
species. The extensive study allows us to suggest with confidence,
Please cite this article in press as: de Souza, T.O., et al., Population structure and identification of two matrilinear and one patrilinear
mitochondrial lineages in the mussel Mytella charruana, Estuarine, Coastal and Shelf Science (2014), http://dx.doi.org/10.1016/j.ecss.2014.11.009
T.O. de Souza et al. / Estuarine, Coastal and Shelf Science xxx (2014) 1e10
the origin in time of the three distinct lineages (haplogroups) of COI
sequences. Two were matrilinear, separated from each other
probably around 3.4 and 4 Mya, and one patrilinear, which split
from the matrilinear lineage around 51.2 Mya, before the separation
of the genera Perna and Mytella. The correct identification of COI
haplogroups and their haplotypes in charru mussels now allow
these sequences to be used with confidence for taxonomic purposes and in management for aquaculture and conservation. Only
haplogroup A is found around the Amazon river discharge, suggesting that there is some environmental factor in that area
selecting against individuals from haplogroup B. Differences in
salinity and temperature may have selected for distinct lineages of
mussels (Sirna-Terranova et al., 2006) and more detailed data on
temperature, pH, salinity and local currents could help explain the
genetic structuring observed among populations of Brazilian
Mytella charruana. Global warming is changing the coast line and
changing the freshwater inflow into estuaries that may cause severe bottlenecks effects, especially in estuarine species (Roessig
et al., 2004). Knowledge of the genetic diversity of the species
and their tolerance to environmental changes is necessary and
urgent to plan strategies to minimize local impacts of global
warming, especially when a considerable number of the involved
species are used as a food source, such as fishes and mussels.
Acknowledgments
We thank the Conselho Nacional de Desenvolvimento Científico
gico (CNPq) for funding this research via a grant (Edital
e Tecnolo
Universal 2009, Grant No. 472558/2009-9) and a scholarship
(552344/2010-9) awarded to T. Souza. Sampling was carried out
under License No. 21823-1 from the Instituto Chico Mendes de
~o da Biodiversidade (ICMBIO). We would like to thank
Conservaça
Mauro Melo and Jaqueline Oliveira for collecting some of the
samples used in this paper.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ecss.2014.11.009.
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