Lack of introgressive hybridization by North African catfish (Clarias gariepinus) in native Vietnamese bighead catfish (Clarias macrocephalus) populations as revealed by novel nuclear and mitochondrial markers

Accepted Manuscript Lack of introgressive hybridization by North African catfish (Clarias gariepinus) in native Vietnamese bighead catfish (Clarias macrocephalus) populations as revealed by novel nuclear and mitochondrial markers

Thuy-Yen Duong, Kim T. Scribner, Jeannette Kanefsky, Uthairat Na-Nakorn PII: DOI: Reference:

S0044-8486(16)30560-9 doi: 10.1016/j.aquaculture.2017.03.007 AQUA 632554

To appear in:

aquaculture

Received date: Revised date: Accepted date:

12 October 2016 18 January 2017 1 March 2017

Please cite this article as: Thuy-Yen Duong, Kim T. Scribner, Jeannette Kanefsky, Uthairat Na-Nakorn , Lack of introgressive hybridization by North African catfish (Clarias gariepinus) in native Vietnamese bighead catfish (Clarias macrocephalus) populations as revealed by novel nuclear and mitochondrial markers. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Aqua(2016), doi: 10.1016/j.aquaculture.2017.03.007

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Lack of introgressive hybridization by North African catfish (Clarias gariepinus) in native Vietnamese bighead catfish (Clarias macrocephalus) populations as revealed by novel nuclear and mitochondrial markers

PT

Authors: Thuy-Yen Duonga,*, Kim T. Scribnerb, c, Jeannette Kanefskyb, and Uthairat Na-

RI

Nakornd

College of Aquaculture and Fisheries, Can Tho University, Viet Nam

b

Department of Fisheries and Wildlife, Michigan State University, East Lansing, MI 48824,

NU

a

SC

Address:

c

MA

USA

Department of Integrative Biology, Michigan State University, East Lansing, MI 48824,

USA

Kasetsart University, 50 Ngamvongwan Rd., Chatujak, Bangkok 10900, Thailand

*

Corresponding author: Thuy-Yen Duong, College of Aquaculture and Fisheries, Can Tho

AC CE P

TE

D

d

University, 3/2 street, Can Tho City, Viet Nam. Fax: +84 710 3830323 E-mail: [email protected]

1

ACCEPTED MANUSCRIPT

ABSTRACT The widespread farming of catfish (genus Clarias) hybrids between bighead catfish (C. macrocephalus, Cm) and the introduced North African Catfish (C. gariepinus, Cg) in the Mekong Delta, where flooding is common, has raised concerns of introgression of Cg into

PT

the native catfish due to backcrossing by escaped hybrids with wild Cm. This study employed novel PCR-RFLP analyses of a mitochondrial (Cytochrome c oxidase subunit I, COI) and two

RI

nuclear (Rhodopsin and Tropomyosin) markers and six microsatellite loci to differentiate

SC

hybrid individuals from their parental species, and evaluate whether introgression has occurred in wild and cultured Cm populations in the region. Results of marker screening

NU

showed that sizes and frequencies of microsatellite alleles differed between the two parental

MA

species. Four loci exhibited fixed species-specific differences in allele frequency (monomorphic in Cg and highly polymorphic in Cm). Levels of sequence variation varied at

D

the three genes examined, ranging from 3 to 8% (sequence length 652 bp for COI, 802 bp for

TE

Rhodopsin and 925 bp for Tropomyosin). Based on variable sites identified that were not shared between the two species, 3 restriction enzymes (SpeI, XcmI and PflMI) were selected

AC CE P

to digest PCR products at species-specific sites within the COI, Rhodopsin and Tropomyosin genes, respectively. Results of PCR-RFLP analyses confirmed that Cm is the maternal lineage of cultured hybrids, while nuclear genes (Rhodopsin and Tropomyosin) and microsatellite loci revealed that hybrids possessed admixed multi-locus genotypes relative to the two parental species. Analyses of six microsatellite loci and PCR-RFLP of two nuclear genes in 473 individuals collected from 11 wild and three cultured populations revealed one F1 hybrid in the wild but no evidence of widespread introgression into native bighead catfish. These results differ from findings in Thailand where intensive catfish culture and deliberate species hybridization is practiced.

2

ACCEPTED MANUSCRIPT

AC CE P

TE

D

MA

NU

SC

RI

PT

Keywords: introgression, hybrid identification, Clarias, PCR-RFLP, microsatellite, mtDNA

3

ACCEPTED MANUSCRIPT

1. Introduction Introductions of fish species have occurred frequently around the globe, both intentionally and accidentally (FAO, 1997; Gozlan et al., 2010). Intentional translocation of fish for aquacultural purposes has been the main cause of non-native species range expansion

PT

(Allendorf et al., 2001; Naylor et al., 2001; Scribner et al., 2001; Senanan et al., 2004). Although non-native species have contributed substantially to aquacultural production (De

RI

Silva et al., 2009; Naylor et al., 2001), their use has also raised serious concerns that non-

SC

indigenous species may hybridize with native species. If hybrids can backcross with parental species, introgression could potentially lead to the extinction of native species (Na-Nakorn et

NU

al., 2004; Pérez et al., 2003; Rhymer and Simberloff, 1996). The introduced Nile tilapia

MA

Oreochromis niloticus, for example, hybridizes with two native Oreochromis species, O. andersonii and O. macrochir, in the Kafue River, Zambia, causing negative impacts on the

D

genetic resources for future aquaculture and commercial fisheries (Deines et al., 2014). In the

TE

U.K., native crucian carp Carassius carassius also faced severe ecological and genetic threats through direct competition and hybridization with introduced goldfish Carassius auratus and

AC CE P

common carp Cyprinus carpio which were released into the wild. As a result in the release of non-native species, hybrids have been found in 38% of British crucian carp populations (N = 21). In some locales, pure crucian carp were not caught due to high abundance and competition abilities of hybrids (Hanfling et al., 2005). Problems associated with introgression can become more serious when hybrids are artificially produced for aquaculture. One such example is the hybrid produced by crossing introduced North African catfish (Clarias gariepinus; Cg) and bighead catfish (C. macrocephalus; Cm), native to Southeast Asia. Cg was introduced to Southeast Asian countries around the 1970s (FAO, 1997). Cg males have been widely hybridized with Cm

4

ACCEPTED MANUSCRIPT

females to produce hybrids for aquaculture (Teugels et al., 1998). Their hybrids have been considered one of the most successful inter-specific hybridizations used in aquaculture (NaNakorn, 2013). They have been cultured widely in Thailand (Bartley et al., 2001; Na-Nakorn et al., 2004) and Viet Nam (Legendre and Antoine, 1998). The widespread farming of hybrids

PT

in areas where flooding occurs in the rainy season (e.g., the Chao Phraya River basin in Central Thailand and Lower Mekong River basin in Viet Nam) has led to hybrids escaping

RI

into the wild, which has been reported in Thailand (Na-Nakorn et al., 2004; Senanan et al.,

SC

2004) and Viet Nam (by 100 % Vietnamese fish farmers interviewed, N = 173, Duong et al., in press). Given that escapement by hybrids has been ongoing for several decades of hybrid

NU

farming, introgression is predicted to occur ubiquitously in wild populations.

MA

Concerns about the potential for widespread introgressive hybridization in wild Vietnamese Cm populations is motivated by knowledge of releases of Cm*Cg hybrids and by

D

previous studies reporting evidence of introgression of African catfish into the native bighead

TE

catfish in Thailand. Na-Nakorn et al. (2004) reported that allozyme alleles of the North African catfish were found in 12 of 25 wild populations and one hatchery population of

AC CE P

bighead catfish in Thailand. Another study in Thailand used six allozyme loci and one microsatellite locus and confirmed evidence of Cg alleles in Cm populations (Senanan et al., 2004). Senanan et al. (2004) found one F1 hybrid and 21 hybrids of higher filial generation (1% to 11% of sampled individuals, total N = 515) which carried one or two diagnostic African catfish allozyme alleles from 4 wild and 2 cultured populations in central Thailand. Both studies suggested that introgression has occurred for several generations in different hybrid catfish farming areas in Thailand. Findings from previous studies in Thailand have raised concerns of widespread genetic introgressive hybridization in wild bighead catfish populations in Viet Nam, particularly in

5

ACCEPTED MANUSCRIPT

the lower Mekong Delta (MD), where hybrids between bighead and African catfish have been cultured since 1990, and where flooding commonly compromises the integrity of aquaculture facilities along major rivers. Hybrid farms are mainly located in several provinces along the Mekong River, which produced 13,000 tons in 2014 (Duong et al., in press). Flooding occurs

PT

annually in the region during the rainy season from August to November. Extensive hybrid farming and annual seasonal flooding in drainages supporting the regional aquaculture

RI

industry in Viet Nam is similar to that in Thailand. If introgressive hybridization mediated by

SC

backcrossing of F1 Cm*Cg to wild Cm is also happening in Viet Nam, additional and more powerful markers and statistical approaches are required to characterize the situation in wild

NU

populations.

MA

Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and microsatellite analyses are effective and simple DNA-based methods that are commonly used

D

to identify inter-specific hybrids (Scribner et al., 2001). PCR-RFLP analyses of mitochondrial

TE

or nuclear genes use restriction enzymes that cut DNA at species-diagnostic sites. The RFLP patterns for mitochondrial DNA in hybrids reflect the maternal species due to maternal

AC CE P

inheritance. RFLP patterns for nuclear genes represent one allele from each parent species (Hashimoto et al., 2010). This method has been used successfully in identifying hybrids, for example, the hybrids between Leporinus macrocephalus and Leporinus elongatus (Hashimoto et al., 2010), and Pseudoplatystoma corruscans and P. reticulatum (Vaini et al., 2014) in Brazil. Microsatellite markers are co-dominant and hybrids possess alleles of both species. Microsatellites have been used in hybridization and introgression studies in many taxa including birds (Hansson et al., 2012) and fish (Aboim et al., 2010; Allendorf et al., 2001; Scribner et al., 2001). The combined use of microsatellite and PCR-RFLP markers in analyses will increase the accuracy of hybrid identification and will provide insights into rates and

6

ACCEPTED MANUSCRIPT

direction of introgression. Although allozyme markers have been used in previous studies on hybridization/introgression between these two species, DNA markers are better suited for this purpose due to their higher levels of polymorphism and also for practical reasons such as the stringent conditions needed for sampling and preservation of tissue for allozyme analysis (Liu

PT

and Cordes, 2004). The objectives of this study were (i) to develop species-diagnostic molecular markers

RI

(microsatellite and PCR-RFLP of mitochondrial and nuclear genes) to distinguish these two

SC

Clarias species and identify their hybrids; (ii) to determine whether introgression of African catfish has occurred into native bighead catfish C. macrocephalus throughout the Mekong

NU

Delta, Viet Nam.

MA

2. Materials and methods 2.1. Fish sampling

D

Samples of sub-adult and adult bighead catfish (weight range 36 – 327 g) were collected

TE

from 11 wild populations and three populations at domesticated culture facilities (total N = 473) in the Mekong Delta, Viet Nam (Fig. 1, Table 1). North African catfish (502 – 1,680 g)

AC CE P

and Clarias hybrids (168 – 340 g) were sampled in a hatchery located in Chau Thanh District, Hau Giang province (9°55′18” N, 105°48′26” E). Fish were transferred alive to a laboratory at Can Tho University, where fish species were classified morphologically based on Rainboth (1996) and Tran et al., (2013). The hybrids are characterized by slightly yellowish coloration (similar to bighead catfish) and a triangular occipital process. A piece of fin (~ 1 cm2) from each sample was collected and preserved in 95% ethanol for genetic analyses. The bighead catfish samples (N = 37) used for PCR-RFLP testing and microsatellite screening were from conservation areas of Long An (Lang Sen Wetland Reserve), Ca Mau (U-Minh Ha) and Kien Giang (U-Minh Thuong) provinces where no catfish or hybrid catfish

7

ACCEPTED MANUSCRIPT

farming has been practiced. These samples were used as references for hybrid testing and identification of introgressed individuals collected from different populations. 2.2. DNA extraction, microsatellite PCR and genotyping Genomic DNA was extracted from fin clips using the QIAGEN DNeasy Blood and

PT

Tissue kit(R) (QIAGEN, Inc.). Concentration and quality of DNA extracts were measured using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Inc.). DNA was then

RI

diluted to 20 ng/µL for PCR.

SC

Microsatellite markers from previous studies (Galbusera et al., 1996; Nazia and Azizah, 2014; Sukkorntong et al., 2008; Yue et al., 2003) were screened. Six microsatellite loci that

NU

amplified in both Cg and Cm species and with different allele sizes were selected for analysis

MA

(Table 2). PCR conditions included 1 X buffer, 1.5 mM MgCl2, 0.2 mM dNTPs, 0.5 µM each forward (fluorescently labeled for microsatellite primers) and reverse primer, 1.25 U Taq (Taq

D

DNA Polymerase, InvitrogenTM, Life Technologies Inc.), and 100 ng DNA template per 25

TE

µL total PCR volume. PCR cycles were based on references (Table 2) with some modifications of the annealing temperature and time and the number of cycles.

AC CE P

Six microsatellite loci were genotyped for reference samples (including 37 Cm, 29 Cg and 30 hybrid samples) using an ABI PRISM® 3130 Genetic Analyzer at the Michigan State University Research Technology Support Facility (MSU RTSF). Allele sizes were used as standards for further genotyping of all samples of cultured and wild bighead catfish by using 6% polyacrylamide gels and a Hitachi FMBIO II scanner. 2.3. PCR-RFLP of mitochondrial and nuclear genes 2.3.1. PCR and sequencing of three genes One mitochondrial gene (Cytochrome c oxidase subunit I, COI) and two nuclear genes (Rhodopsin and Tropomyosin) were amplified using previously described primers (Table 2).

8

ACCEPTED MANUSCRIPT

Restriction endonuclease digestion of mitochondrial COI PCR products was used to determine the maternal origin, and thus the direction of hybridization of commercially cultured catfish. Restriction endo-nuclease digestion of PCR products for the two nuclear genes was employed as co-dominant markers to differentiate hybrids from their parental

PT

species. PCR conditions were as described in the original references. To find sequence differences between the two species (Cm and Cg) in order to search

RI

for species-specific restriction sites, the PCR products of the three genes (COI, Rhodopsin and

SC

Tropomyosin) were cleaned using a QIAquick PCR Purification Kit (QIAGEN, Inc.) and then sequenced using an ABI 3730 DNA Analyzer (conducted by the MSU RTSF). Primers for 2-

NU

directional sequencing of COI are M13F (5’-TGTAAAACGACGGCCAGT-3’) and M13R

MA

(5’-CAGGAAACAGCTATGAC-3’) (Ivanova et al., 2007), and those used for Rhodopsin and Tropomyosin are the forward and reverse primers used in the amplification reactions. The

D

number of samples sequenced depended on the data available in GENBANK. For COI, 99

TE

sequences were available (23 for Cm and 73 for Cg. We sequenced 6 samples for each species. For Rhodopsin and Tropomyosin, no sequences of either species have been reported

AC CE P

except 6 Rhodopsin sequences for Cg. We, therefore, sequenced 12 samples for each gene and for each species. Sequences for the three genes for each species were submitted to GENBANK.

2.3.2. Sequence alignment and selection of restriction enzymes Sequences were aligned using ClustalW implemented in the program MEGA 6.0 (Tamura et al., 2013). After alignment, nucleotide composition, the number of site differences and the Kimura 2-parameter genetic distances within and between the two catfish species were estimated for each gene. Kimura 2-parameter values were chosen for estimation of genetic distances based on the evaluations of molecular model fit implemented in MEGA 6.0.

9

ACCEPTED MANUSCRIPT

The two models Tamura -3 parameter and Kimura 2-parameter were the best models with the lowest BIC (Bayesian information criterion) values. Both models gave the same results of genetic distances within and between species. The aligned sequences (652 bp of COI gene, 802 bp of Rhodopsin, and 923 bp of Tropomyosin) were used to search for restriction sites at sequences

by

using

Restrictionmapper

(available

at

PT

conserved

http://www.restrictionmapper.org/). Enzymes were selected based on the following criteria:

RI

(i) There was only one restriction site, which is species-specific (cuts in only one species); (ii)

SC

restriction sites are at positions not less than 100 bp from the two ends so that fragments can be visualized easily using ethidium bromide stain and 1% agarose gels.

NU

2.3.3. Enzyme incubation and RFLP electrophoresis

MA

PCR products of COI, Rhodopsin and Tropomyosin were incubated separately using the restriction enzymes SpeI, XcmI and PflMI (New England Biolabs, 10 U/µL), respectively. The total reaction volume of 12 µL contained, 5 U enzyme and 4 µL PCR product and incubation

TE

D

took place at 37oC for 150 minutes. Restriction fragments were separated on a 1.4% agarose gel. Fragment sizes were estimated based on a 100 bp DNA ladder (InvitrogenTM) and

software.

AC CE P

visualized and recorded by a UVP gel documentation system and Doc-ITLS image analysis

Five samples of each fish group (Cm, Cg and hybrid) were first tested with PCR-RFLP procedures mentioned above. The results were then applied to identify hybrids from all bighead catfish samples collected. 2.4 Microsatellite data analysis Allele frequencies for each species were estimated using GenAlEX 6.5 (Peakall and Smouse, 2012). Differences in allele and genotype frequencies between species were tested using a Fisher’s exact test in program GENEPOP 4.0 (Raymond and Rousset, 1995; Rousset, 2008).

10

ACCEPTED MANUSCRIPT

Program GENPOP was also used to estimate variance in allele frequency between species (Fst; Weir and Cockerham, 1984) and its analog Rst, which incorporates allele sizes (Rousset, 1996; Slatkin, 1995). Principal Coordinates Analysis (PCoA) were estimated based on the pairwise genetic distance between individuals, using GenAlex 6.5. We also used

PT

STRUCTURE program to estimate posterior probabilities of species origin to identify hybrids from the dataset of 473 bighead catfish, 29 Cg and 37 hybrid individuals. We used the

RI

admixture ancestry model and assumed correlated allele frequencies: 5,000 replicates were

SC

used as a Burn-in period and 50,000 replicates were used to estimate posterior probabilities of species assignment assuming two groups (K = 2; Cm and Cg). The program STRUCTURE

NU

does not explicitly test alternative hypotheses of individual classification best supported based

MA

on estimates of posterior probabilities of species assignment, nor can statistical evaluations be made to determine whether individuals with intermediate posterior probabilities of species

D

assignment are hybrids. Accordingly, analyses were also performed using the Bayesian

TE

Analysis of Population Structure (BAPS) program (Corander et al., 2004) version 4.0. We utilized the non-spatial genetic mixture analysis of BAPS to assign individuals to Cm, Cg or

AC CE P

hybrid groups using an admixture analysis (Corander and Marttinen, 2006) to document evidence of, and statistical support for introgression (individuals of admixed ancestral origins with ancestral contributions from members of different species). 3. Results

3.1. Sequence differences and RFLP of three genes of bighead- and North American catfish, and their hybrids Comparing COI sequences (652 bp) between the two Clarias species, 83 variable nucleotide sites were found, resulting in a Kimura 2-parameter genetic distance between species of 0.16 ± 0.019, which is higher than within genetic distance among samples of each

11

ACCEPTED MANUSCRIPT

species (Table 3). All individuals of each species sequenced were monomorphic at the two nuclear loci Rhodopsin and Tropomyosin. The two species differed at 22 variable sites (out of 802 bp) in the Rhodopsin gene. For the Tropomyosin gene region sequenced, the Cm sequences were 847 bp in length while the Cg sequences were 925 bp in length, due to length

PT

variation in an intron present in this region. If the Tropomyosin length difference is disregarded, there are 51 polymorphic sites between Cg and Cm. Based on the sequence

RI

variation observed, restriction enzyme sites specific for each species were identified within

SC

each of the three genes.

Three enzymes were selected to cut COI, Rhodopsin, and Tropomyosin within

NU

conserved sequences of one species (see Table 3). SpeI was selected for cutting the Cg COI

MA

sequence at the recognition size 5’-A CTAGT-3’, resulting in two fragments of approximately 230 and 500 bp (sizes of PCR products approximately 730 bp). Digestion with SpeI showed

D

that the COI sequence of Cm and the hybrid was not cut and hence resulted in a single band of

TE

approximately 730 bp, meanwhile, Cg had 2 bands of the predicted sizes of 230 and 500 bp (Fig. 2). The result confirmed that the hybrids possessed Cm mitochondrial DNA.

AC CE P

For the Rhodopsin gene, restriction enzyme XcmI cut Cm only was chosen, producing 2 fragments with similar sizes, 420 and 460 - 480 bp. Variation in sizes of these two bands depends on the effectiveness of amplification at the two ends of the Rhodopsin gene. Rhodopsin products digested by XcmI showed different sized bands in Cm and Cg, in which one band is of approximately 900 bp in Cg, 2 bands have similar sizes of 420 and 480 bp in Cm. The hybrids had 3 bands, one of approximately 900 bp and two bands of 420 and 480 bp, representing for both parental species (Fig. 3). Tropomyosin PCR products for Cg and Cm differed by approximately 100 bp (approximately 900 and 1000 bp, respectively). Digestion of the Tropomyosin PCR product

12

ACCEPTED MANUSCRIPT

with PflMI cut only for Cg, producing two bands of approximately 420 and 580 bp for Cg samples while one band (undigested) for Cm PCR products. Tropomyosin gene PCR patterns in hybrids showed three bands characteristic of both parents (Fig. 4). 3.2. Differences in microsatellite genotypes between bighead and North African catfish and

PT

their hybrids Four out of six microsatellite markers (Cm02, Cm05, Cb19, and NCmD11) exhibited

RI

fixed species-specific differences in allele sizes and did not overlap allele size ranges. Each

SC

was monomorphic in Cg and highly polymorphic in Cm (Table 4). The two species shared one common allele at the other two loci (Cg6 and Cg9) but exhibited significant differences in

NU

allele frequencies (multi-locus estimates of Fst = 0.525 and Rst = 0.95; P<0.01). Genotypes of

MA

hybrids included alleles inherited from both parental species. 3.3. Testing for species introgression using PCR-RFLP and microsatellite loci

D

PCR-RFLP procedures were applied to 473 bighead catfish samples. Results showed

TE

that only one sample collected from a rice field in Tra Vinh province had F1 hybrid patterns of two nuclear genes of Rhodopsin and Tropomyosin (Cm pattern in COI gene), whereas all

AC CE P

other (N = 472 samples) had Cm RFLP patterns in all three genes. The results from the six microsatellite markers confirmed the PCR-RFLP results. One hybrid individual identified from PCR-RFLP methods was also concluded to be an F1 hybrid due to heterozygous genotypes at all six loci. Meanwhile, the other samples (N = 472) were concluded to be bighead catfish because of species-specific alleles at four microsatellite loci (Appendix, Table A.1). Twenty-one Cm samples had one allele of Cg6 locus (size 132 bp), other 7 samples had one allele of Cg9 (size 182 bp), and one sample had two alleles (Cg6-132 and Cg9 -182), which are similar to allele sizes of Cg (with frequencies of 0.845 for Cg6-132 and 0.534 for Cg9 -182). These 30 samples were distributed across 13 of 14 populations

13

ACCEPTED MANUSCRIPT

(except a cultured population in Hau Giang, No. 9 in Table 1 and Fig. 1). The principal coordinate analysis showed that all 472 Cm samples formed in one group which was divergent from Cg and the hybrid (Fig. 5). This main difference was observed in the first coordinate which explained 12.76% of the microsatellite variation among samples.

PT

3.4. Posterior probabilities of species assignment The admixture analysis implemented in program STRUCTURE based on six

RI

microsatellite loci indicated that the posterior probability of Cm and Cg being assigned to

SC

their respective species was 0.997, while that of the F1 hybrid assigned to with intermediate posterior probability to both species ranged 0.487 and 0.513 to Cg and Cm, respectively. All

NU

know hybrid control individuals and the one F1 hybrid from a wild population were

MA

confirmed as hybrids (P<0.01) based on posterior probabilities of admixture vs pure species hypothesis testing in program BAPS. Twenty-eight individuals that had one to two alleles that

D

were found commonly in Cg but not observed in Cm but were assigned to Cm with high

with two alleles).

AC CE P

4. Discussion

TE

posterior probabilities (≥0.984 for individuals with one allele, and 0.932 for one individual

The important contributions from this study are the identification of markers (PCRRFLP and microsatellite) that distinguish Clarias catfish hybrids from their parent species, and the lack of widespread evidence of introgression of African catfish genes in native bighead catfish in the Mekong Delta, Viet Nam. 4.1. DNA markers for distinguishing Clarias species and their hybrids Uncertainty associated with the morphological classification of hybrids is one of the most basic difficulties in investigations of inter-specific hybridization and introgression (Bohling et al., 2013). DNA-based methods have been used to increase the accuracy in hybrid

14

ACCEPTED MANUSCRIPT

detection (Hashimoto et al., 2010). In the present study, the use of data based on PCR-RFLP analysis of mitochondrial and nuclear genes identified maternal lineages of each hybrid and their parental species. The production of a single fragment of the same size in Cm and the cultured hybrid from digestion of the COI gene (mtDNA) with SpeI confirms that Cm is the

PT

maternal lineage of the cultured hybrids in Viet Nam. This direction of hybridization is consistent with commercial hybrid farming practices in Thailand (Na-Nakorn et al., 2004;

RI

Senanan et al., 2004).

SC

Applications of PCR-RFLP profiles from nuclear genes in fish is feasible when universal primers are available for amplification of genes (such as Rhodopsin, recombination

NU

activating genes RAG1 and RAG2, Tropomyosin) used in previous studies (Chen et al., 2008;

MA

Kochzius et al., 2010; Larmuseau et al., 2010; López et al., 2004). Different genes have different substitution rates (Zhang et al., 2002) which also vary among taxa (Shen et al.,

D

2013). Among nuclear genes, Rhodopsin is one of the most taxonomically divergent genes.

TE

Chen et al (2008) found that Rhodopsin in two species representing two genera (Leuciscinae and Rasborinae) of the family Cyprinidae showed high substitution rates (resulting in genetic

AC CE P

distance between two species of 0.160) compared to other nuclear genes (0.081 – 0.139). In the sand goby family Gobiidae, Rhodopsin was also found to have high variation (average genetic distance 0.11) among 4 genera (Larmuseau et al., 2010). In our study, the genetic divergence of Rhodopsin sequences between two species in the genus Clarias was 0.027, comparable to genetic distances found between species within other genera, such as Danio, Puntius and Devario (family Cyprinidae), which range from 0.002 to 0.041 (Collins et al., 2012). Variation in Rhodopsin sequences between Cm and Cg and highly conserved segregating sites within species indicated that this gene is reliable for catfish hybrid identification.

15

ACCEPTED MANUSCRIPT

Tropomyosin is an -nuclear gene that has been used in previous studies of other Clarias species (Hashimoto et al., 2011, 2010) and Characiform fishes (Calcagnotto et al., 2005). We found that this gene is highly variable in terms of the number of species informative sites and sequence length differences between the two Clarias species but highly

PT

conserved within each species (all 12 sequences of each species are monomorphic). Together with the PCR-RFLP polymorphisms, the microsatellite markers used in this

RI

study allow accurate identification of hybrids and evidence of introgression (Bohling et al.,

SC

2013; Epifanio and Philipp, 1997). In some cases, the use of microsatellites alone is difficult to differentiate hybrids due to high levels of polymorphism (Toniato et al., 2010). Taking

NU

advantages of microsatellite markers available for different Clarias species, we selected loci

MA

that amplified in both species, but where the species were characterized by fixed differences (N = 4 loci) or differences in allele frequency (N = 2 loci; Fst = 0.525, and Rst = 0.95) with

D

minimal overlap in allele size distributions. In addition, the combination of two markers and

TE

two approaches can be complementary and provide stronger evidence. 4.2. Introgression in bighead catfish

AC CE P

Results based on inspection of genotypes at eight loci (six microsatellite loci and two PCR-RFLP nuclear loci) and admixture analysis (based on six microsatellite loci) indicate one F1 hybrid out of all wild individuals sampled. Except for the single F1 hybrid, the other 472 wild and cultured bighead catfish samples were identified as pure Cm. According to equations provided by Epifanio and Philipp (1997), when eight species-specific co-dominant loci were used, the probability of error of misidentifying backcrossed individuals as a pure bighead catfish is 0.4% (or 0.5L where L is the number of loci used, equation 2), and that of misidentifying an F2 hybrid as a pure Cm is 1.5 x 10-5 (or 0.52L, equation 1). Therefore, it is unlikely that bighead catfish samples were misclassified. Moreover, F2 and other higher

16

ACCEPTED MANUSCRIPT

generation hybrids might not be present in the wild because F2 progeny were not successfully produced in direct F1 experimental crosses (Nukwan et al., 1990, cited by Na-Nakorn et al., 2004) nor could they survive after the fingerling stages (Liem, 2008). However, further empirical studies should be conducted to test these findings.

PT

Our results showed 30 Cm individuals (6.36% of investigated samples, N = 472) possess one or two (in one individual) microsatellite alleles that have high frequencies in Cg.

RI

This phenomenon can be explained by three hypotheses: (i) past introgression, (ii) homoplasy

SC

(convergence due to mutation), or (iii) symplesiomorphy or co-segregation of share alleles from a common ancestor (Avise and Saunders, 1984). The third hypothesis could be excluded

NU

given the high level of sequence divergence (for three genes, especially COI gene in this

MA

study) between African and bighead catfish and their widely dispersed native ranges, it is unlikely that these species would share alleles due to common ancestry. Data on cytochrome b

D

also confirmed high genetic divergence between the two species (Agnese and Teugels, 2005).

TE

Under the first hypothesis, low frequencies of shared alleles in Cm at two out of eight

AC CE P

loci (both PCR-RFLP and microsatellite loci) could be a consequence of backcrossing of hybrids with members of native Cm populations. F1 catfish hybrids can be partially fertile with different levels of gonad abnormalities (12.5% in female N = 43, and 8.2% in male N = 52) under artificial propagation conditions (Liem, 2008). Liem (2008) reported that offspring produced by backcrossing F1 hybrids with male Cm was unsuccessful with almost zero fertilization rate (1.32 ±1.12%), whereas backcrossing F1 hybrids with female Cm yielded a higher fertilization rate (58.03 ±14.17%), hatching rate of 45.48±7.03% and normal fry of 41±4.91%. Liem (2008) reported that backcrossed individuals had gonad abnormalities in 35% of females (N = 43), and 6.52% of males (N = 49). Mature (10 – 12 months old) males produced no sperm. Despite limited empirical work on backcrossing of catfish hybrids, the

17

ACCEPTED MANUSCRIPT

study by Liem (2008) implied that backcrossing of F1 hybrids and advanced generations with Cm females could be possible but postzygotic reproductive isolation could prevent or limit the spread of higher filial generation backcrosses. In contrast to previous studies in wild Thailand populations based on allozyme markers, they found evidence for low levels of introgression

PT

(Na-Nakorn et al., 2004; Senanan et al., 2004). Evidence of introgression in Vietnamese bighead catfish populations is not supported.

RI

First, the distribution of 30 individuals that carry Cg microsatellite alleles seems to be random

SC

among 13 populations (except one cultured population in Hau Giang province; Fig. 1), of which four populations (3, 4, 12, and 14 in Table 1) were collected in the protected areas (i.e.

NU

conservation parks) where hybrid farming has not been practiced in large surrounding areas

MA

and the whole provinces. If introgression had occurred, the number of introgressed individuals would likely be more commonly found near hybrid farming and flooding areas (provinces

D

along Mekong River). In addition, given that hybrid farming is still popular in Viet Nam and

TE

the escape of hybrids is ongoing, backcrossing F1 hybrids with female Cm that happened in the past would be expected to be an ongoing process and many more F1 hybrids would be

AC CE P

expected. However, we found all tested individuals were pure Cm with a low probability of misclassification (0.4%) of F1 backcrosses based on data from six microsatellite and two PCR-RFLP markers. To increase assignment accuracy of backcrosses or individuals of higher filial generations, use of many additional loci is required (Vähä and Primmer, 2006). The second hypothesis (homoplasy) or conversion of allele sizes in the 2 species may explain our results of low frequencies of Cg microsatellite alleles in wild Cm populations. Alleles at Cg6 and Cg9 loci overlap in one portion of the size range or alleles between the two species (Cm and Cg) with low frequencies in Cm and high frequencies in Cg (Appendix, Table A.1). The presence of Cg alleles within the size range of Cm populations could be a

18

ACCEPTED MANUSCRIPT

result of high microsatellite mutation rates. Patterns of allele size and frequencies in Cm (combined with hybrids) could be explained by the stepwise mutation model where mutations change a single repeat (Ohta and Kimura, 1973). At Cg6 locus, for example, the overlap in sizes between the two species range from 128 bp to 134 bp, in which frequency of allele 132

PT

is 0.022 in Cm and 0.845 in Cg, whereas, hybrids possess four alleles in this range (128 – 134), indicating that Cm could each hold each allele size from 130 and above. There was also

RI

evidence of two-phase mutation model found in microsatellite loci with more than one steps

SC

of mutation changes (Di Rienzo et al., 1994). In addition, mutational patterns vary among microsatellite loci and among species at the same loci (Estoup et al., 2002; Takezako and Nei,

NU

1996). Our data are consistent with the homoplasy hypothesis. It could explain why sharing

MA

alleles between the two Clarias species has been observed in two loci but not the others, and allele distributions in almost all Cm populations regardless the proximity of hybrid farming.

D

These Cg6 and Cg9 primers also yielded the same size PCR products in other species of

1996).

TE

family Clariidae (C. anguillaris, C. alluaudi, and Heterobranchus longifilis (Galbusera et al.,

AC CE P

Karyological analyses also provide a possible explanation why backcrossing of catfish hybrids with purebred Cm individuals could be limited. The karyotype of Cg is 2n = 56 (Teugels et al., 1992), and that of Cm is 2n = 52 (Sittikraiwong, 1987). Cm x Cg hybrids have intermediate numbers of chromosomes (2n = 54; (Visoottiviseth et al., 1997). Similarly, hybrids between Clarias gariepinus and Heterobranchus longifilis (2n = 52) also have 2n = 54 chromosomes (Teugels et al., 1992). 4.3. Implications and future research Accurate identification of hybrid catfish individuals is important in both aquaculture and fisheries management globally given the importance of catfish in global aquaculture

19

ACCEPTED MANUSCRIPT

within and beyond native species distributional boundaries. In aquaculture, use of hybrid catfish as parents can result in slow growth and reduced disease resistance (Senanan et al., 2004). In fisheries, management of aquatic genetic resources has been challenging due to the escape of hybrids or introduced species into the wild (Allendorf et al., 2001; Scribner et al.,

PT

2001). Therefore, easily applied DNA-based methods such as PCR-RFLP analyses of maternally inherited mitochondrial COI and bi-parentally inherited nuclear genes to identify

RI

hybrid individuals provide valuable tools in aquaculture and fisheries management of Clarias

SC

and can be applied to other species.

Concerns about genetic contamination arising from hybrid catfish farming, a sector with

NU

an important contribution to inland aquaculture production in Asia (De Silva et al., 2009) are

MA

widespread, including in Viet Nam. Concerns have been raised in Viet Nam given the widespread use of hybrids in aquaculture and high potential for accidental release in the lower

D

Mekong River, which is one of the last strongholds of viable natural populations of bighead

TE

catfish in the region. It is, therefore, significant that introgression has not occurred widely in bighead catfish in Viet Nam. However, additional cytological and ecological empirical

AC CE P

experiments should be conducted to identify which of several pre- and post-zygotic species isolating mechanisms are involved in preventing introgression. Moreover, findings from this study raise the question why there is a lack of evidence of introgression in Viet Nam when the situation in Thailand is different. The difference may relate with much larger quantity of escaped or released (due to religious belief) hybrids into wild habitats in Thailand than in Viet Nam. The larger annual production in Thailand (in the period 1990 – 2002, hybrid production was 32,000 - 64,000 tons: FAO, 2016) may have led to more escapes. Strain effects between bighead catfish in Thailand and Viet Nam could be also another possible reason leading to differences in hybridization outcomes (Dunham, 2011). These hypotheses need further

20

ACCEPTED MANUSCRIPT

research. Moreover, future research that focuses on causal relationships between successful or unsuccessful proliferation of hybrids in the wild would inform fish culture globally to minimize negative effects on native wild populations. Acknowledgements

PT

This research is funded by the Vietnam National Foundation for Science and Technology Development (NAFOSTED) under grant number: 106-NN.05-2014.86. The first author thanks

RI

Vietnam Education Foundation for supporting the Visiting Scholar grant 2014, for which she could

SC

work on genetic analyses at the Molecular Ecology Genetic Lab, Department of Fisheries and Wildlife, Michigan State University.

NU

References

Aboim, M.A., Mavárez, J., Bernatchez, L., Coelho, M.M., 2010. Introgressive hybridization

MA

between two Iberian endemic cyprinid fish: A comparison between two independent hybrid zones. J. Evol. Biol. 23, 817–828. doi:10.1111/j.1420-9101.2010.01953.x

D

Allendorf, F.W., Leary, R.F., Spruell, P., Wenburg, J.K., 2001. The problems with hybrids:

AC CE P

5347(01)02290-X

TE

Setting conservation guidelines. Trends Ecol. Evol. 16, 613–622. doi:10.1016/S0169-

Avise, J.C., Saunders, N.C., 1984. Hybridization and introgression among species of sunfish (Lepomis): analysis by mitochondrial DNA and allozyme markers. Genetics 108, 237– 255.

Bartley, D.M., Rana, K., Immink, A. J., 2001. The use of inter-specfic hybrid in aquaculture and fisheries. Rev. Fish Biol. Fish. 10, 325–337. Bohling, J.H., Adams, J.R., Waits, L.P., 2013. Evaluating the ability of Bayesian clustering methods to detect hybridization and introgression using an empirical red wolf data set. Mol. Ecol. 22, 74–86. doi:10.1111/mec.12109 Calcagnotto, D., Schaefer, S. A., DeSalle, R., 2005. Relationships among Characiform fishes 21

ACCEPTED MANUSCRIPT

inferred from analysis of nuclear and mitochondrial gene sequences. Mol. Phylogenet. Evol. 36, 135–153. doi:10.1016/j.ympev.2005.01.004 Chen, W.-J., Miya, M., Saitoh, K., Mayden, R.L., 2008. Phylogenetic utility of two existing and four novel nuclear gene loci in reconstructing Tree of Life of ray-finned fishes: the

PT

order Cypriniformes (Ostariophysi) as a case study. Gene 423, 125–34. doi:10.1016/j.gene.2008.07.016

RI

Chen, W.-J., Ortí, G., Meyer, A., 2004. Novel evolutionary relationship among four fish

SC

model systems. Trends Genet. 20, 424–31. doi:10.1016/j.tig.2004.07.005 Chen, W.J., Bonillo, C., Lecointre, G., 2003. Repeatability of clades as a criterion of

NU

reliability: A case study for molecular phylogeny of Acanthomorpha (Teleostei) with

MA

larger number of taxa. Mol. Phylogenet. Evol. 26, 262–288. doi:10.1016/S10557903(02)00371-8

D

Collins, R.A., Armstrong, K.F., Meier, R., Yi, Y., Brown, S.D.J., Cruickshank, R.H., Keeling,

TE

S., Johnston, C., 2012. Barcoding and border biosecurity: Identifying cyprinid fishes in the aquarium trade. PLoS One 7. doi:10.1371/journal.pone.0028381

AC CE P

Corander, J., Marttinen, P., 2006. Bayesian identification of admixture events using multilocus molecular markers. Mol. Ecol. 15, 2833–2843. doi:10.1111/j.1365294X.2006.02994.x

Corander, J., Waldmann, P., Marttinen, P., Sillanpää, M.J., 2004. BAPS 2: Enhanced possibilities for the analysis of genetic population structure. Bioinformatics 20, 2363– 2369. doi:10.1093/bioinformatics/bth250 De Silva, S.S., Nguyen, T.T.T., Turchini, G.M., Amarasinghe, U.S., Abery, N.W., 2009. Alien species in aquaculture and biodiversity: a paradox in food production. Ambio. 38, 24–28. doi:doi:10.1579/0044-7447-38.1.24

22

ACCEPTED MANUSCRIPT

Deines, A.M., Bbole, I., Katongo, C., Feder, J.L., Lodge, D.M., 2014. Hybridisation between native Oreochromis species and introduced Nile tilapia O. niloticus in the Kafue River, Zambia. African J. Aquat. Sci. 39, 23–34. doi:Doi 10.2989/16085914.2013.864965 Di Rienzo, A., Peterson, A.C., Garza, J.C., Valdes, A.M., Slatkin, M., Freimer, N.B., 1994.

Acad. Sci. 91, 3166–3170. doi:10.1073/pnas.91.8.3166

PT

Mutational processes of simple-sequence repeat loci in human populations. Proc. Natl.

RI

Dunham, R., 2011. Aquaculture and fisheries biotechnology: genetic approaches, 2nd ed.

SC

CABI Publishing.

Duong TY, Cau N.V, Long, DN, in press. The development history of hybrid catfish farming

NU

in the Mekong delta and the perception of farmers on hybrid issues. Journal of Science,

MA

Can Tho University, Viet Nam

Epifanio, J.M., Philipp, D.P., 1997. Sources for misclassifying genealogical origins in mixed

D

hybrid populations. J. Hered. 88, 62–65. doi:10.1093/oxfordjournals.jhered.a023059

TE

Estoup, A., Jarne, P., Cornuet, J.M., 2002. Homoplasy and mutation model at microsatellite loci and their consequences for population genetics analysis. Mol. Ecol. 11, 1591–1604.

AC CE P

doi:10.1046/j.1365-294X.2002.01576.x FAO, 1997. FAO database on introduced aquatic species. FAO Database Introd. Aquat. Species, FAO, Rome.

FAO, 2016. Global Aquaculture Production Database 1950–2014. Rome, Italy. http://www.fao.org/fishery/statistics/global-aquaculture-production, 25 September 2016. Fitzgibbon, J., Hope, A., Slobodyanyuk, S.J., Bellingham, J., Bowmaker, J.K., Hunt, D.M., 1995. The rhodopsin-encoding gene of bony fish lacks introns. Gene 164, 273–277. doi:10.1016/0378-1119(95)00458-I Friesen, V.L., Congdon, B.C., Kidd, M.G., Birt, T.P., 1999. Polymerase chain reaction (PCR)

23

ACCEPTED MANUSCRIPT

primers for the amplification of five nuclear introns in vertebrates. Mol. Ecol. 8, 2147– 2149. doi:10.1046/j.1365-294X.1999.00802-4.x Galbusera, P., Volckaert, F. A, Hellemans, B., Ollevier, F., 1996. Isolation and characterization of microsatellite markers in the African catfish Clarias gariepinus

PT

(Burchell, 1822). Mol. Ecol. 5, 703–705. Gozlan, R.E., Britton, J.R., Cowx, I., Copp, G.H., 2010. Current knowledge on non-native

RI

freshwater fish introductions. J. Fish Biol. 76, 751–786. doi:10.1111/j.1095-

SC

8649.2010.02566.x

Hanfling, B., Bolton, P., Harley, M., Carvalho, G.R., 2005. A molecular approach to detect

NU

hybridisation between crucian carp (Carassius carassius) and non-indigenous carp

MA

species (Carassius spp. and Cyprinus carpio). Freshw. Biol. 50, 403–417. doi:DOI 10.1111/j.1365-2427.2004.01330.x

D

Hansson, B., Tarka, M., Dawson, D.A., Horsburgh, G.J., 2012. Hybridization but no evidence

TE

for backcrossing and introgression in a sympatric population of great reed warblers and clamorous reed warblers. PLoS One 7. doi:10.1371/journal.pone.0031667

AC CE P

Hashimoto, D.T., Mendonça, F.F., Senhorini, J.A., Bortolozzi, J., de Oliveira, C., Foresti, F., Porto-Foresti, F., 2010. Identification of hybrids between Neotropical fish Leporinus macrocephalus and Leporinus elongatus by PCR-RFLP and multiplex-PCR: Tools for genetic monitoring in aquaculture. Aquaculture 298, 346–349. doi:10.1016/j.aquaculture.2009.11.015 Hashimoto, D.T., Mendonça, F.F., Senhorini, J.A., de Oliveira, C., Foresti, F., Porto-Foresti, F., 2011. Molecular diagnostic methods for identifying Serrasalmid fish (Pacu, Pirapitinga, and Tambaqui) and their hybrids in the Brazilian aquaculture industry. Aquaculture 321, 49–53. doi:10.1016/j.aquaculture.2011.08.018

24

ACCEPTED MANUSCRIPT

Ivanova, N. V., Zemlak, T.S., Hanner, R.H., Hebert, P.D.N., 2007. Universal primer cocktails for fish DNA barcoding. Mol. Ecol. Notes 7, 544–548. doi:10.1111/j.14718286.2007.01748.x Kochzius, M., Seidel, C., Antoniou, A., Botla, S.K., Campo, D., Cariani, A., Vazquez, E.G.,

PT

Hauschild, J., Hervet, C., Hjörleifsdottir, S., Hreggvidsson, G., Kappel, K., Landi, M., Magoulas, A., Marteinsson, V., Nölte, M., Planes, S., Tinti, F., Turan, C., Venugopal,

RI

M.N., Weber, H., Blohm, D., 2010. Identifying fishes through DNA barcodes and

SC

microarrays. PLoS One 5, 1–15. doi:10.1371/journal.pone.0012620 Larmuseau, M.H.D., Huyse, T., Vancampenhout, K., Van Houdt, J.K.J., Volckaert, F.A.M.,

NU

2010. High molecular diversity in the rhodopsin gene in closely related goby fishes: A

doi:10.1016/j.ympev.2009.10.007

MA

role for visual pigments in adaptive speciation? Mol. Phylogenet. Evol. 55, 689–98.

D

Legendre, M., Antoine, P. (eds)., 1998. The biological diversity and aquaculture of Clariid

TE

and Pangasiid catfishes in South-East Asia: Proceedings of the mid-term workshop of the “Catfish Asia project”. Jakarta (IDN); Can Tho : IRD ; Can Tho.

AC CE P

Liem, P.T., 2008. Breeding performance and traits inheritance of hybrid catfish, Clarias macrocephalus and Clarias gariepinus. Ph.D dissertation, Universiti Malaysia Terengganu, Malaysia.

Liu, Z.J., Cordes, J.F., 2004. DNA marker technologies and their applications in aquaculture genetics. Aquaculture 238, 1–37. doi:10.1016/j.aquaculture.2004.05.027 López, J.A., Chen, W.-J., Ortí, G., 2004. Esociform phylogeny. Copeia. doi:10.1643/CG-03087R1 Na-Nakorn, U., 2013. Interspecific Hybrid Catfish in Thailand, in: Ruane, J., Dargie, J.., Mba, C., Boettcher, P., Makkar, H.P.., Bartley, D.., Sonnino, A. (Eds.), Biotechnologies at

25

ACCEPTED MANUSCRIPT

work for smallholders: Case studies from developing countries in crops, livestock and fish. FAO, Rome, pp. 149–155. Na-Nakorn, U., Kamonrat, W., Ngamsiri, T., 2004. Genetic diversity of walking catfish, Clarias macrocephalus, in Thailand and evidence of genetic introgression from

PT

introduced farmed C. gariepinus. Aquaculture 240, 145–163. doi:10.1016/j.aquaculture.2004.08.001

RI

Naylor, R., Williams, S.L., Strong, D.R., 2001. Aquaculture - A gateway for exotic species.

SC

Science 294, 1655–1666. doi:10.1126/science.1064875

Nazia, A.K., Azizah, M.N.S., 2014. Isolation of microsatellites in the bighead catfish, Clarias

NU

macrocephalus and cross-amplification in selected Clarias species. Mol. Biol. Rep. 41,

MA

1207–1213. doi:DOI 10.1007/s11033-013-2965-9

Ohta, T., Kimura, M., 1973. A model of mutation appropriate to estimate the number of

D

electrophoretically detectable alleles in a finite population. Genet. Res. 22, 201–204.

TE

doi:10.1017/S0016672300012994

Peakall, R., Smouse, P.E., 2012. GenALEx 6.5: Genetic analysis in Excel. Population genetic

AC CE P

software for teaching and research-an update. Bioinformatics 28, 2537–2539. doi:10.1093/bioinformatics/bts460 Pérez, J.E., Alfonsi, C., Nirchio, M., Munoz, C., Gómez, J.A., 2003. The introduction of exotic species in aquaculture: A solution or part of the problem? Interciencia 28, 234– 238.

Rainboth, W.J., 1996. Fishes of the Cambodian Mekong. FAO, Rome, Italy, 265p. Raymond, M., Rousset, F., 1995. GENEPOP (version 1.2): population genetics software for exact tests and ecumenicism. J. Hered. 86, 248–249. Rhymer, J.M., Simberloff, D., 1996. Extinction by hybridization and introgression. Annu.

26

ACCEPTED MANUSCRIPT

Rev. Ecol. Syst. 27, 83–109. doi:10.1146/annurev.ecolsys.27.1.83 Rousset, F., 2008. GENEPOP’007: A complete re-implementation of the GENEPOP software for Windows and Linux. Mol. Ecol. Resour. 8, 103–106. doi:10.1111/j.14718286.2007.01931.x

PT

Rousset, F., 1996. Equilibrium values of measures of population subdivision for stepwise mutation processes. Genetics 142, 1357–1362.

RI

Scribner, K.T., Page, K.S., Bartron, M.L., 2001. Hybridization in freshwater species: a review

SC

of case studies and cytonuclear methods of biological inference. Rev. Fish Biol. Fish. 10, 293–323.

NU

Senanan, W., Kapuscinski, A.R., Na-Nakorn, U., Miller, L.M., 2004. Genetic impacts of

MA

hybrid catfish farming (Clarias macrocephalus x C. gariepinus) on native catfish populations in Central Thailand. Aquaculture 235, 167–184.

D

doi:10.1016/j.aquaculture.2003.08.020

TE

Shen, X.X., Liang, D., Feng, Y.J., Chen, M.Y., Zhang, P., 2013. A versatile and highly efficient toolkit including 102 nuclear markers for vertebrate phylogenomics, tested by

AC CE P

resolving the higher level relationships of the Caudata. Mol. Biol. Evol. 30, 2235–2248. doi:10.1093/molbev/mst122 Sittikraiwong, P., 1987. Karyotype of the hybrid between Clarias macrocephalus Gunther and Pangasius sutchi Fowler. M.Sc. Thesis. Fac. Fish. Kasetsart Univ. Thailand. Slatkin, M., 1995. A measure of population subdivision based on microsatellite allele frequencies. Genetics 139, 457–462. Sukkorntong, C., Panprommin, D., Poompuang, S., 2008. Sixteen EST-linked microsatellite markers in Gunther’s walking catfish, Clarias macrocephalus. Mol. Ecol. Resour. 8, 1300–1302. doi:10.1111/j.1755-0998.2008.02165.x

27

ACCEPTED MANUSCRIPT

Takezako, N., Nei, M., 1996. Genetic distances and reconstruction of phylogenetic trees from microsatellite DNA. Genetics 399, 389–399. doi:10.1016/0006-3207(96)85988-X Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA6: Molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30, 2725–2729.

PT

doi:10.1093/molbev/mst197 Teugels, G.G., Legendre, M., Le, T.H., 1998. Preliminary results on the morphological

RI

characterisation of natural populations and cultured strains of Clarias species

SC

(Siluriformes, Clariidae) from Vietnam. Biol. Divers. Aquac. Clariid Pangasiid catfishes South-East Asia. Proc. mid-term Work. Catfish Asia Project. 27–30.

NU

Teugels, G.G., Ozouf-Costza, C., Legendre, M., Parrent, M., 1992. A karyological analysis of

MA

the artificial hybridization between Clarias gariepinus (Burchell, 1822) and Heterobranchus longifilis Valenciennes, 1840 (Pisces; Clariidae). J. Fish Biol. 40, 81–

D

86. doi:10.1111/j.1095-8649.1992.tb02555.x

TE

Toniato, J., Penman, D.J., Martins, C., 2010. Discrimination of tilapia species of the genera Oreochromis, Tilapia and Sarotherodon by PCR-RFLP of 5S rDNA. Aquac. Res. 41,

AC CE P

934–938. doi:10.1111/j.1365-2109.2009.02366.x Tran, D.D., Shibukawa, K., Nguyen, T.P., Ha, P.H., Tran, X.L., Mai, V.H., Utsugi, K., 2013. Fishes of the Mekong Delta, Viet Nam. Cantho University Publishing House. Vähä, J.P., Primmer, C.R., 2006. Efficiency of model-based Bayesian methods for detecting hybrid individuals under different hybridization scenarios and with different numbers of loci. Mol. Ecol. 15, 63–72. doi:10.1111/j.1365-294X.2005.02773.x Vaini, J.O., Grisolia, A.B., do Prado, F.D., Porto-Foresti, F., 2014. Genetic identification of interspecific hybrid of Neotropical catfish species (Pseudoplatystoma corruscans vs. Pseudoplatystoma reticulatum) in rivers of Mato Grosso do Sul State, Brazil. Neotrop.

28

ACCEPTED MANUSCRIPT

Ichthyol. 12, 635–641. doi:Doi 10.1590/1982-0224-20130169 Visoottiviseth, P., Sungpetch, A., Pongthana, N., 1997. Karyotype of hybrid catfish (Clarias macrocephalus x C. gariepinus). J. Sci. Soc. Thail. 24, 57–63. Ward, R.D., Zemlak, T.S., Innes, B.H., Last, P.R., Hebert, P.D.N., 2005. DNA barcoding

PT

Australia’s fish species. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 360, 1847–1857. doi:10.1098/rstb.2005.1716

RI

Weir, B.S., Cockerham, C.C., 1984. Estimating F-statistics for the analysis of population

SC

structure. Evolution (N. Y). 38, 1358–1370. doi:10.2307/2408641

Yue, G.H., Kovacs, B., Orban, L., 2003. Microsatellites from Clarias batrachus and their

MA

doi:10.1046/j.1471-8286.2003.00486.x

NU

polymorphism in seven additional catfish species. Mol. Ecol. Notes 3, 465–468.

Zhang, L., Vision, T.J., Gaut, B.S., 2002. Patterns of nucleotide substitution among

D

simultaneously duplicated gene pairs in Arabidopsis thaliana. Mol. Biol. Evol. 19, 1464–

AC CE P

TE

1473.

29

ACCEPTED MANUSCRIPT

Table 1 Sampling locations and population characteristics (wild/cultured) of bighead catfish samples No.

Location (province)

Wild

or Latitude

Longtitude

size

Tan Hung (Long An)

Cultured

10°55'27.9"N

2.

Chau Doc (An Giang)

Wild

10°40'59.9"N

105°04'54.0"E

26

3.

Tam Nong (Dong Thap)*

Wild

10°39'57.9"N

105°33'53.4"E

36

4.

Lang Sen (Long An)*

Wild

10°46'31.2"N

105°42'38.2"E

53

5.

An Huu (Tien Giang)

Wild

10°18'43.4"N

105°53'34.8"E

36

6.

Tan Ngai (Vinh Long)

Wild

10°15'44.7"N

105°55'15.2"E

28

7.

Omon (Can Tho)

Cultured

10°08'53.5"N

105°36'04.2"E

40

8.

Phong Dien (Can Tho)

Wild

10°00'37.4"N

105°38'41.8"E

43

9.

Chau Thanh A (Hau Giang)

Cultured

9°55'26.4"N

105°43'23.0"E

26

Wild

9°43'58.7"N

106°03'27.6"E

22

Wild

9°47'55.4"N

106°24'12.8"E

17

12. U-Minh Thuong (Kien Giang)*

Wild

9°39'17.6"N

105°06'41.6"E

31

13. Vinh Loi (Bac Lieu)

Wild

9°20'22.4"N

105°43'58.5"E

30

Wild

9°19'28.9"N

104°53'20.9"E

45

AC CE P

TE

11. Cau Ngang (Tra Vinh)

14. U-Minh Ha (Ca Mau)*

RI

NU

MA D

10. Dai Ngai (Soc Trang)

105°33'16.5"E

PT

1.

SC

Cultured

Sample

(*) Fish were sampled from conservation areas

30

40

ACCEPTED MANUSCRIPT

Table 2 Primer sequences for PCR of sequenced genes (COI, Rhodopsin, and Tropomyosin) and microsatellite loci Primer name

Sequence 5’ – 3’

Ta*

References

COI

Fish F2-t1

TGTAAAACGACGGCCAGTCGACT

52

Ivanova et al.,

al., 2005

CAGGAAACAGCTATGACACTTCA

SC

Fish R2-t1

2007; Ward et

RI

AATCATAAAGATATCGGCAC

PT

Gene

GGGTGACCGAAGAATCAGAA

RH1039-R

TGCTTGTTCATGCAGATGTAGA

Trop-F

GAGTTGGATCGGGCTCAGGAGCG

Trop-R

Cga09

Cba19

Cma02

F

1999

TE

AC CE P

Cga06

Friesen et al.,

CGGTCAGCCTCTTCAGCAATGTGC TT

Microsatellite primers

Chen et al., 2003

NU

CNTATGAATAYCCTCAGTACTACC

MA

Tropomyosin

RH193-F

D

Rhodopsin

CAGCTCGTGTTTAATTTGGC

55

Galbusera et al., 1996

R

TTGTACGAGAACCGTGCCAGG

F

CGTCCACTTCCCCTAGAGCG

57

Galbusera et al., 1996

R

CCAGCTGCATTACCATACATGG

F

CAGGGCTAAATTACCCATAATCA

R

GGCATGTGTTATAACATGTGAGG

F

GAGCAATCAGCAGTGGAG

31

59

Yue et al., 2003

59

Sukkorntong et

ACCEPTED MANUSCRIPT

al., 2008

Cma05

R

AGGCAACAGTGAAACAGC

F

GAGATGACGTGTGTAGCAC

53

Sukkorntong et al., 2008

GACCTGACTTTCAGGAAGC

F

ACCACTGGAGCACGCATATC

50

Nazia and

PT

NCmD11

R

R

RI

Azizah, 2014

GTTTCGAATTATAGGGCGGAGAG

AC CE P

TE

D

MA

NU

SC

(*) Ta = Annealing temperature after PCR optimization

32

ACCEPTED MANUSCRIPT

Table 3 DNA fragments of COI, Rhodopsin, and Tropomyosin genes and RFLP enzymes selected Description

COI

Rhodopsin

Tropomyosin

Approximate length of amplified

800

900

1000 for Cg 900 for Cm

652

83

Between –species genetic

0.16 ± 0.019

NU

Numbers of variable sites

difference**

SpeI

MA

Selected Restriction enzyme

Cg

Species cut by selected enzymes

925 for Cg 847 for Cm

22

51 (+78)*

0.027 ± 0.006

0.062 ± 0.008

XcmI

PflMI

Cm

Cg

SC

fragment after alignment (bp)

802

RI

Length of trimmed DNA

PT

DNA fragment (bp)

D

*Tropomyosin differs at 51 variable sites and is 78 bp shorter in Cm compared to Cg.

TE

**Based on Kimura 2-parameter method. Within-group genetic differences was found only in

AC CE P

the mitochondrial COI gene, 0.010± 0.002 for Cg and 0.007 ± 0.002 for Cm.

33

ACCEPTED MANUSCRIPT

Table 4 Sizes (bp) and numbers (#) of alleles and measures of genetic diversity for C. macrocephalus (Cm), C. gariepinus (Cg), and their hybrids based on 6 microsatellite loci Loci Size

Cg (n = 29)

238-264

# alleles

Hybrid (n = 30) 209

PT

Cm02

Cm (n = 37)

1

11

211

211 - 236

1

6

222

220 - 262

1

12

225

223 - 300

21

1

18

132 – 168

128-132

128 -168

17

2

16

164-182

182-190

172-190

7

2

7

12.67 (2.14)

1.33 (0.21)

11.67 (1.94)

8.89 (2.06)

1.22 (0.16)

5.36 (0.79)

0.766 (0.061)

0.126 (0.082)

0.956 (0.029)

uHe

0.847 (0.054)

0.129 (0.087)

0.795 (0.026)

Fis

0.083 (0.043)

- 0.042 (0.082)

- 0.205 (0.044)

# alleles NCmD11

9 244 – 264

Size

13

NU

# alleles

254 – 300

Size

MA

Cb19

# alleles Size

D

Cg6

Size

AC CE P

# alleles

TE

# alleles Cg9

RI

224 – 244

Size

SC

Cm05

14

209-264

Measures of genetic diversity Na Ne Ho

Note: Na: Number of alleles; Ne: Effective number of alleles; Ho: Observed Heterozygosity, uHe: Unbiased Expected Heterozygosity; and Fis: Fixation Index, Fis = (He-Ho)/He.

34

ACCEPTED MANUSCRIPT

Appendix Table A.1: Allele sizes and frequencies of bighead catfish (Cm), North American catfish (Cg), and the hybrids at six microsatellite loci Allele size

Cm

Cg

Hybrid

Cm05

N

472

29

30

0.015

226

0.272

228

0.274

230

0.127

232

0.044

AC CE P

RI

470

128

NU

224

N

29 0.155

130 132

Hybrid 30 0.100 0.217

0.022

0.845

0.133

0.150

134

0.133

136

0.015

0.117

138

0.097

140

0.084

0.017

MA

0.003

0.417

TE

222

Cg6

Cg

SC

0 1.000

D

211

Cm

PT

Locus

0.117

234

0.165

0.167

142

0.059

0.017

236

0.025

0.017

144

0.003

0.033

238

0.037

146

240

0.012

148

0.218

0.100

242

0.014

150

0.084

0.017

244

0.008

152

0.024

0.017

35

0.017

ACCEPTED MANUSCRIPT

472

29

220 1.000 0.003

242

0.029

244

0.023

246

0.007

248

0.061

250

0.072

252

0.109

254

0.017

30

156

0.090

0.233

158

0.097

0.067

0.267

160

0.062

0.033

0.017

162

0.046

0.017

164 166

0.010

168

0.009

0.017

170

0.014

0.033

176

0.002

0.083 Cg9

N

472

0.199

0.067

164

0.001

256

0.127

0.050

168

0.001

258

0.127

0.117

172

0.008

0.017

260

0.119

0.050

174

0.122

0.100

262

0.072

0.050

176

0.125

264

0.038

178

0.125

0.150

266

0.005

180

0.608

0.333

268

0.003

182

0.008

AC CE P

TE

D

NU

0.017

0.022

MA

240

0.043

RI

222

154

PT

NCmD11 N

0.002

SC

246

36

0.083

29

0.534

30

0.067

ACCEPTED MANUSCRIPT

0.003

184

272

0.001

188 29

30

223

190

0.067 Cm02 N

225

1.000

256

0.011

258

0.010

260

0.005

262

0.006

264

238

0.466 470

29 1.000

0.233 30 0.500

0.038

240

0.074

242

0.072

244

0.145

0.050

246

0.123

0.133

248

0.110

0.017

0.017

250

0.098

0.067

0.031

0.017

252

0.086

0.050

266

0.010

0.017

254

0.073

268

0.016

0.033

256

0.062

0.067

270

0.033

258

0.055

0.050

272

0.040

0.067

260

0.036

274

0.032

0.033

262

0.011

0.033

276

0.043

0.033

264

0.014

0.017

NU

0.014

0.017

MA

254

209

D

0.015

TE

250

0.417

SC

227

0.001

PT

470

0.100

RI

N

AC CE P

Cb19

270

37

0.017

ACCEPTED MANUSCRIPT

0.036

0.017

266

0.001

280

0.044

0.033

268

0.001

282

0.072

284

0.068

286

0.098

0.067

288

0.061

0.033

290

0.141

0.033

292

0.097

0.033

294

0.053

296

0.030

298

0.026

300

0.010

MA

NU

SC

RI

PT

278

0.050

AC CE P

TE

D

0.017

38

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

D

Fig. 1. The Mekong Delta (Viet Nam) with 11 wild (circles) and three (triangles; 1, 7, and 9)

TE

cultured populations of bighead catfish Clarias macrocephalus. The number of 14

AC CE P

populations corresponds to population information presented in Table 1. The asterisk in the wild population 11 (Tra Vinh) indicates one F1 hybrid found, inferred from cytonuclear data.

39

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

Fig. 2. PCR-RFLP patterns of COI gene digested with enzyme SpeI. Lanes 1: 100 bp ladder;

MA

2-5: C. macrocephalus (Cm); 6-9: C. gariepinus (Cg); 10-13: the hybrids; 14-16 undigested

AC CE P

TE

D

COI of Cm, Cg, and the hybrid, respectively.

40

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

Fig. 3. PCR-RFLP patterns of Rhodopsin gene digested with enzyme XcmI. Lanes 1: 100 bp

MA

ladder; 2-5: C. macrocephalus (Cm); 6-9: C. gariepinus (Cg); 10-13: the hybrids; 14-16

AC CE P

TE

D

undigested Rhodopsin of Cm, Cg, and the hybrid, respectively.

41

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

Fig. 4. PCR-RFLP patterns of Tropomyocin gene digested with enzyme PflMI. Lanes 1: 100

MA

bp ladder; 2-5: C. macrocephalus (Cm); 6-9: C. gariepinus (Cg); 10-13: the hybrids; 14-16

AC CE P

TE

D

undigested Tropomyocin of Cm, Cg, and the hybrid, respectively.

42

Coord. 2 (5.66 explained variation)

ACCEPTED MANUSCRIPT

PT

Cg

Hybrid

NU

SC

RI

Cm

Coord. 1 (12.76 explained variation)

MA

Fig. 5. Principal coordinate analysis based on six microsatellite loci of C. macrocephalus

AC CE P

TE

D

(Cm), C. gariepinus (Cg), and the hybrid

43

ACCEPTED MANUSCRIPT

Highlights of the manuscript

- Six microsatellites and PCR-RFLP of three genes were developed to accurately identify hybrids between Clarias macrocephalus and C. gariepinus. - Microsatellite and PCR-RFLP data showed that 6.36% of individuals from 13 Vietnamese bighead populations carried one or two microsatellite alleles found in North African catfish is

PT

likely due to homoplasy or convergent mutation of allele sizes in the two species, rather than historical genetic introgression.

RI

- Findings of lack of evidence of genetic introgression in Viet Nam differs from previous

SC

studies in Thailand, leading to interesting areas for further research directed toward reduced

AC CE P

TE

D

MA

NU

threats of genetic contamination to native populations in areas of intensive aquaculture.

44