- Email: [email protected]
Mitochondrial DNA evidence for late Pleistocene population expansion of the white catfish, Ameiurus catus
Biochemical Systematics and Ecology 42 (2012) 94–98
Contents lists available at SciVerse ScienceDirect
Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco
Mitochondrial DNA evidence for late Pleistocene population expansion of the white catfish, Ameiurus catus Abinash Padhi* Department of Biological Science, University of Tulsa, 800 S. Tucker Drive Tulsa, OK-74104, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 31 August 2011 Accepted 18 February 2012 Available online 22 March 2012
Employing distance-based, maximum likelihood-based, and Bayesian coalescent methodologies and utilizing mitochondrial control region sequence data, the present study reports the importance of Pleistocene glacial epochs on the demographic expansion of the white catfish, Ameiurus catus. White catfish are endemic to the southeastern region of the United States, the region that had never glaciated during the Pleistocene epoch; however, compared to the present day meandering river patterns, the region did exhibit braided river patterns during the late Quaternary. The present study revealed the existence of two distinct matrilineal lineages of white catfish, southeast Gulf coast (SEGC) and southeast Atlantic coastal plains (SEACP), which are estimated to have diverged from their common ancestor approximately 0.71 (0.37–1.1) million years ago. Consistent with the results of the mismatch distribution and site frequency spectrum based summary statistics such as Fu’s F, the Bayesian skyline plot (BSP) has also showed evidence for population expansion. The BSP showed two phase growth: a static phase until 0.2 million years, followed by a sudden population expansion thereafter, which is the time that coincides with the late Quaternary epoch. The changing patterns in drainage systems in response to the Quaternary climate change might have had a significant impact on the demographic expansion of white catfish. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: White catfish mtDNA Genetic diversity Bayesian skyline Quaternary period Demographic history
1. Introduction The Pleistocene glacial epoch played a dominant role influencing the diversity and distribution of several aquatic flora and fauna endemic to the southeastern United States (Avise, 2000; Soltis et al., 2006). The repeated glacial cycles during the late Pleistocene epoch also had a profound influence on the drainage patterns of the southeast Atlantic coastal plain (SEACP) of the United States (Leigh et al., 2004). For instance, in contrast to the present day meandering river patterns, the SEACP region mostly exhibited braided river patterns from the late Quaternary to the Holocene period (Leigh et al., 2004). These changing patterns in drainage systems in response to the Quaternary climate change might have had a profound influence, as well, on the genetic structuring of aquatic species that inhabited the SEACP region. White catfish (Ameiurus catus) of the family Ictaluridae, which inhabits the freshwater and slightly brackish water drainages along the SEACP and west on the Gulf slope to western Alabama of the USA (Page and Burr, 1991; Schultz, 2003; NatureServe, 2006), might possess the footprints of Quaternary climate change on their mitochondrial DNA. This omnivorous, slow-growing fish is relatively smaller in size with average length 25–35 cm (Schultz, 2003) and attains sexual maturity at 3–4 years. Spawning occurs during spring and early summer when the water temperature reaches approximately 20–21 C. While most of the species within the genus Ameiurus have wide distribution across the drainages of the USA, the A. catus native range is restricted to the SEACP region (Page and Burr, 1991). Fossil based
* Present address: Department of Biology, 208 Mueller Laboratory, The Pennsylvania State University, University Park, PA-16802, USA. E-mail address: [email protected]. 0305-1978/$ – see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2012.02.011
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molecular clocks revealed that A. catus diverged from its common ancestor approximately 16 million years ago, a time that falls within the Miocene era (Hardman and Hardman, 2008; Padhi, 2010). The lack of Miocene-Pliocene fossil evidence of A. catus, however, makes it difficult to predict the geographic range of this species during that time. Nevertheless, the report of late Pleistocene fossil evidence from Vero Beach, Indiana River County, Florida indicates the existence of this species in the SEACP region during that time (Weigel,1963). Although fossil evidence suggests the presence of A. catus in the SEACP region during the Pleistocene, it is difficult to infer the effect of late Quaternary climate change on the population dynamics of the species. The uniparentally inherited, recombination free, and rapidly evolving mitochondrial DNA (mtDNA) has been used widely as a genetic marker to track past population dynamics of vertebrates and invertebrates (Avise, 2000). Using the mtDNA control region (CR) and employing distance-based summary statistics (Fu, 1997), mismatch distribution (Herpending, 1994; Rogers, 1995), parsimony network (Templeton et al., 1995), maximum likelihood (Kuhner et al., 1998) and Bayesian coalescent (Drummond et al., 2005; Drummond and Rambaut, 2007) methodologies, the present study investigates the importance of late Quaternary climate change on the intraspecific diversity and past population dynamics of A. catus.
2. Materials and methods The current distribution and approximate geographic origin of A. catus samples is shown in Fig. 1a. Fin clip tissue from each individual was preserved in 75% ethanol and shipped to the University of Tulsa for genetic analyses. Total DNA was extracted from preserved tissue using the phenol-chloroform method and a portion of mtCR (423 bp) amplified and sequenced using the previously described protocols (Murphy and Collier, 1996). Primers: CR-F: 50 -AACTCTCACCCCTAGCTCCCAAAG-30 and CRR: 50 -CCTGAAGTAGGAACCAGATG-30 (Kocher et al., 1989; Meyer et al., 1990) were used for amplification and sequencing of mtCR. Sequences were aligned using MacClade 4.03 (Maddison and Maddison, 2000). CR haplotypes were identified using Collapse 1.2 (Posada, 2005). Unique haplotypes generated in this study were deposited in GenBank (Accession numbers: EF377366 - EF377387). The appropriate nucleotide substitution model of sequence evolution was selected by the hierarchical likelihood ratio tests (hLRTs) implemented in Modeltest, ver. 3.7 (Posada and Crandall, 1998). A haplotype network was constructed at 95% of connection limit (<9 mutational steps) using TCS 1.18 (Clement et al., 2000). Distance-based mismatch histogram (Rogers and Harpending, 1992; Rogers, 1995; Schneider and Excoffier, 1999), site frequency spectrum based summary statistics such as Fu’s F (Fu, 1997), Bayesian and ML-based coalescent approaches (Kuhner et al., 1998) were utilized to infer the demographic history. Arlequin ver. 3.1 (Schneider et al., 2000) was used to compute the frequency of pairwise differences to evaluate the hypothesis of sudden expansion. Using the same program, Fu’s FS statistic (Fu, 1997) as an indicator of demographic expansion was also estimated. The ML-based growth parameter (g) was estimated using the Markov Chain Monte Carlo (MCMC) method implemented in Fluctuate ver. 1.4 (Kuhner et al., 1998). Ten short MCMCs of 200 generations each and two long chain MCMCs of 20,000 generations were set for each run. The program was run 10 times with different seeds and the mean and standard deviation were estimated from these results of separate runs. Since Fluctuate ver 1.4 may have biased upwards in estimating ‘g’ (Kuhner et al., 1998), a more conservative approach in testing for significance was used (g > 3 g (SD)) (Lessa et al., 2003). Using a rate of 0.015 nucleotide substitutions per site per million year (divergence rate z3% per myr) that was previously estimated for Ictalurus furcatus (Padhi, 2011) as the mean rate, the program BEAST ver 1.48 (Drummond and Rambaut, 2007), which employs a Bayesian MCMC coalescent approach, was used to estimate the time to the most recent common ancestor (TMRCA) and the Bayesian skyline plot (BSP) under a strict clock model with the HKY þ G nucleotide substitution model. The MCMC chains were run for 5 million times to ensure that convergence (allowing a 10% burn-in) was reached. Tracer ver 1.4 was used to check the convergence and to estimate 95% highest posterior density (HPD) of the respective parameters and BSP. The MCMC samples were summarized to infer the maximum clade credibility (MCC) tree using TreeAnnotator v1.4.8 (Drummond and Rambaut, 2007), with posterior probability values shown for the major branches.
3. Results Parsimony network analyses and the MCC tree have revealed the existence of two distinct matrilineal lineages of A. catus (Figs. 1b and 2a). Samples from SEACP formed a unique cluster and are separated from the southeast Gulf coast (SEGC) lineage by five mutational steps (Fig. 1b). Clustering of a few individuals from SEGC with the SEACP clade suggests the possibility of accidental introductions to the SEACP drainage system or the possibility of incomplete lineage sorting. The unimodal pattern of the mismatch distribution of A. catus is best fit to the model of sudden expansion (Fig. 1c). In addition, the significant negative Fs and a significant positive growth parameter (Fig. 1c) further support the hypothesis of sudden expansion of A. catus. Based on the Bayesian coalescent method, SEACP and SEGC lineages are estimated to have diverged from their common ancestor approximately 0.71 (0.37–1.1) million years ago (Fig. 2a) and all the haplotypes appear to have diverged approximately 0.2 myr ago (Fig. 2a), the time that coincides with the late Quaternary epoch. Consistent with the results of mismatch distribution, the BSP has also shown evidence for population expansion. The BSP showed two phase growth: a static phase (phase I) until 0.2 million years ago, followed by a sudden population expansion thereafter (phase II). The timing of haplotype divergence is consistent with the population expansions as revealed by BSP (Fig. 2a and b).
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Fig. 1. Geographic origin of samples, haplotype network, and the mismatch histogram for Ameiurus catus inferred from mtDNA control region sequence data. (a) Map showing geographic origin of samples from Gulf coast and southeast Atlantic coast drainages of USA. 1: Ochlockenee River, Florida (n ¼ 10); 2: Satilla River, Georgia (n ¼ 12); 3: St. Mary River, Florida (n ¼ 11); 4: Savannah River, South Carolina (n ¼ 10), and 5: Roanoke, River, North Carolina (n ¼ 2). (b) Parsimony network analyses showing genealogical relationships among the haplotypes. All the haplotypes were connected at the 95% confident limit. The size of the circle is proportional to the number of individuals. Empty circles are the hypothetical mutational steps. Lines connecting two haplotypes represent one mutational step. Drainages are colour coded. (c) Mismatch histogram showing unimodal distribution is not statistically significant from the model of sudden population expansion. Significant negative Fu’s F and significant positive growth (g) parameter (mean g > 3SD of g) also indicate population expansion.
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Fig. 2. Maximum clade credibility (MCC) tree and Bayesian skyline plot (BSP) showing the timing of haplotype divergence and population dynamics of A. catus. (a) MCC tree shows two distinct lineages representing Gulf coast and SE Atlantic coast. The mean time of divergence (in million years) with 95% highest posterior density (HPD) and the posterior probability (nodal support) for respective nodes are mentioned. Posterior probabilities are in italics. (b) BSP showing two phase growth patterns: a steady growth (phase I) until 0.2 myr followed by sudden growth thereafter (phase II). Genetic diversity measured in terms of effective population size is expressed in logarithmic scale in Y-axis, whereas time in million years (myr) is in X-axis.
4. Discussion Employing distance-based, ML-based, and Bayesian coalescent methodologies and utilizing mitochondrial control region sequence data, the present study reports the importance of the last Ice age on the demographic expansion of white catfish, which is endemic to the southeastern region of the United Sates, the region that never glaciated during the Pleistocene epoch. Although the region never glaciated, the repeated glacial cycles during the last glacial maxima had a profound influence on the drainage patterns of this region (Leigh et al., 2004). Therefore, one might expect that biological entities endemic to this region might possess the footprints of these demographic effects on their mitochondrial DNA. The present study revealed that
98
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A. catus is comprised of two distinct matrilineal lineages, SEGC and SEACP, and that their TMRCA is estimated to be 0.7 myr. Results from mismatch distribution, Fu’s F, ML-based coalescent growth parameters, and BSP have also shown evidence of population expansion of A. catus. More importantly, around 0.2 myr all the haplotypes began diverging and the period is marked as a phase of rapid expansion. As observed in the present study for white catfish, previous studies have also reported that several freshwater fishes endemic to the southeastern region of the USA showed genetic discontinuity between Gulf coast and Atlantic coastal plain drainages leading to two distinct matrilineal lineages (Avise, 2000; Soltis et al., 2006). The results of the present study are also consistent with the notion that the late Pleistocene epoch played a dominant role in intraspecific divergence of several aquatic and terrestrial flora and fauna in the southeastern United States (Bermingham and Avise, 1986; Avise, 2000; Soltis et al., 2006). The rapid population expansion and intraspecific diversity of white catfish, which occurred around 0.2 myr, coincides with the time frame when the SEACP region had mostly exhibited braided river patterns (Leigh et al., 2004). The repeated sea level fluctuations during the last ice age (Haq et al., 1987) have had dramatic consequences in changing the dimensions of drainage patterns. Each geographically isolated drainage population with smaller effective population size could eventually result in accumulation of measurable nucleotide differences due to the impact of genetic drift, which is more pronounced for populations with smaller effective population size. The repeated merging and isolation of drainage patterns is therefore believed to have had significant impact on the redistribution of matrilineal lineages (Avise, 2000). Nevertheless, these changing patterns in drainage systems in response to the Quaternary climate change might have had significant impact on the demographic expansion of white catfish and other aquatic species that inhabited the SEACP region. Acknowledgements I thank Dr. Glen Collier for providing laboratory facilities to carry out this work, Dr. Peggy Hill for thoughtful comments towards improving this manuscript and the fisheries biologists of the respective state departments of natural resources for providing samples: Rich Caleteux (Florida), Don Harrison (Georgia), David Allen (South Carolina), and Kent Nelson (North Carolina). I thank Karen Winans for the technical support in DNA sequencing and The University of Tulsa, Office of Research and Sponsored Programs for providing financial support for this study. References Avise, J.C., 2000. Phylogeography: The History and Formation of Species. Harvard University Press, Cambridge, MA. Bermingham, E., Avise, J.C., 1986. Molecular zoogeography of freshwater fishes in the southeastern United States. Genetics 113, 939–965. Clement, M., Posada, D., Crandall, K.A., 2000. TCS: a computer program to estimate gene genealogies. Mol. Ecol. 9, 1657–1659. Drummond, A.J., Rambaut, A., 2007. BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evol. Biol. 7, 214. Drummond, A.J., Rambaut, A., Shapiro, B., Pybus, O.G., 2005. Bayesian coalescent inference of past population dynamics from molecular sequences. Mol. Biol. Evol. 22, 1185–1192. Fu, Y.X., 1997. Statistical tests of neutrality of mutations against population growth, hitchhiking and background selection. Genetics 147, 915–925. Haq, B., Hardenbol, J., Vail, P., 1987. Chronology of fluctuating sea levels since the Triassic. Science 235, 1156–1167. Hardman, M., Hardman, L.M., 2008. The relative importance of body size and paleoclimatic change as explanatory variables influencing lineage diversification rate: an evolutionary analysis of bullhead catfishes (Siluriformes: Ictaluridae). Syst. Biol. 57, 116–130. Herpending, R.C., 1994. Signature of ancient population growth in a low resolution mitochondrial DNA mismatch distribution. Hum. Biol. 66, 591–600. Kocher, T.D., Thomas, W.K., Meyer, A., Edwards, S.V., Paabo, S., Villablanca, F.X., Wilson, A.C., 1989. Dynamics of mitochondrial DNA evolution in animals: amplification and sequencing with conserved primers. Proc. Natl. Acad. Sci. USA 86, 6196–6200. Kuhner, M., Yamamato, J., Felsenstein, J., 1998. Maximum-likelihood estimation of population growth rates based on the coalescent. Genetics 149, 429–434. Leigh, D.S., Srivastava, P., Brook, G.A., 2004. Late Pleistocene braided rivers of the Atlantic coastal plain, USA. Quaternary Sci. Rev. 23, 65–84. Lessa, E.P., Cook, J.A., Patton, J.L., 2003. Genetic footprints of demographic expansion in North America, but not Amazonia, during the late Quaternary. Proc. Natl. Acad. Sci. USA 100, 10331–10334. Maddison, D.R., Maddison, W.P., 2000. MacClade 4: Analysis of Phylogeny and Character Evolution. Sinauer Associates, Sunderland, MA. Meyer, A., Kocher, T.D., Basasibwaki, P., Wilson, A.C., 1990. Monophyletic origin of Lake Victoria cichlid fishes suggested by mitochondrial DNA sequences. Nature 347, 550–553. Murphy, J.W., Collier, G.E., 1996. Phylogenetic relationships within the aplocheiloid fish genus Rivulus (Cyprinodontiformes, Rivulidae): implications for Caribbean and Central American biogeography. Mol. Biol. Evol. 13, 642–649. NatureServe, 2006. Nature Serve Explorer: An Online Encyclopedia of Life [Web Application]. Version 4.7. NatureServe, Arlington, Virginia.. Available: http:// www.natureserve.org/explorer. Padhi, A., 2010. Mitochondrial DNA sequence revealed contrasting demographic history between the black bullhead (Ameiurus melas) and its cryptic lineage in North America. Aquat. Living Resour. 23, 325–333. Padhi, A., 2011. Climate-driven population dynamics of a North American freshwater fish, Ictalurus furcatus: inference from the mitochondrial control region sequence data. Ecol. Freshw. Fish 20 (2), 263–269. Page, L.M., Burr, B.M., 1991. A Field Guide to Freshwater Fishes of North America North of Mexico. Houghton Mifflin Co., Boston, MA. Posada, D., Crandall, K.A., 1998. Modeltest: testing the model of DNA substitution. Bioinformatics 14, 817–818. Posada, D., 2005. Collapse Ver. 1.2. Available from: http://darwin.uvigo.es/. Rogers, A.R., Harpending, H., 1992. Population growth makes waves in the distribution of pairwise genetic differences. Mol. Biol. Evol. 9, 552–569. Rogers, A.R., 1995. Genetic evidence for a Pleistocene population expansion. Evolution 49, 552–569. Schneider, S., Excoffier, L., 1999. Estimation of demographic parameters from the distribution of pairwise differences when the mutation rates vary among sites: application to human mitochondrial DNA. Genetics 152, 1079–1089. Schneider, S., Roessli, D., Excoffier, L., 2000. Arlequin: a Software for Population Genetics Data Analysis, Ver. 3.0. Genetics and Biometry Laboratory, University of Geneva, Switzerland. Schultz, K., 2003. Ken Schultz’s Field Guide to Freshwater Fish. John Wiley & Sons, Inc, Hoboken, NJ. Soltis, D.E., Morris, A.B., McLachlan, J.S., Manos, P.S., Soltis, P.S., 2006. Comparative phylogeography of unglaciated eastern North America. Mol. Ecol. 15, 4261–4293. Templeton, A.R., Routman, E., Phillips, C.A., 1995. Separating population structure from population history: a cladistic analysis of the geographical distribution of mitochondrial DNA haplotypes in the tiger salamander, Ambystoma tigrinum. Genetics 140, 767–782. Weigel, R.D., 1963. Fossil Vertebrates of Vero. Florida Geological Survey, Florida. Special Publication No. 10, 1–59.
Contents lists available at SciVerse ScienceDirect
Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco
Mitochondrial DNA evidence for late Pleistocene population expansion of the white catfish, Ameiurus catus Abinash Padhi* Department of Biological Science, University of Tulsa, 800 S. Tucker Drive Tulsa, OK-74104, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 31 August 2011 Accepted 18 February 2012 Available online 22 March 2012
Employing distance-based, maximum likelihood-based, and Bayesian coalescent methodologies and utilizing mitochondrial control region sequence data, the present study reports the importance of Pleistocene glacial epochs on the demographic expansion of the white catfish, Ameiurus catus. White catfish are endemic to the southeastern region of the United States, the region that had never glaciated during the Pleistocene epoch; however, compared to the present day meandering river patterns, the region did exhibit braided river patterns during the late Quaternary. The present study revealed the existence of two distinct matrilineal lineages of white catfish, southeast Gulf coast (SEGC) and southeast Atlantic coastal plains (SEACP), which are estimated to have diverged from their common ancestor approximately 0.71 (0.37–1.1) million years ago. Consistent with the results of the mismatch distribution and site frequency spectrum based summary statistics such as Fu’s F, the Bayesian skyline plot (BSP) has also showed evidence for population expansion. The BSP showed two phase growth: a static phase until 0.2 million years, followed by a sudden population expansion thereafter, which is the time that coincides with the late Quaternary epoch. The changing patterns in drainage systems in response to the Quaternary climate change might have had a significant impact on the demographic expansion of white catfish. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: White catfish mtDNA Genetic diversity Bayesian skyline Quaternary period Demographic history
1. Introduction The Pleistocene glacial epoch played a dominant role influencing the diversity and distribution of several aquatic flora and fauna endemic to the southeastern United States (Avise, 2000; Soltis et al., 2006). The repeated glacial cycles during the late Pleistocene epoch also had a profound influence on the drainage patterns of the southeast Atlantic coastal plain (SEACP) of the United States (Leigh et al., 2004). For instance, in contrast to the present day meandering river patterns, the SEACP region mostly exhibited braided river patterns from the late Quaternary to the Holocene period (Leigh et al., 2004). These changing patterns in drainage systems in response to the Quaternary climate change might have had a profound influence, as well, on the genetic structuring of aquatic species that inhabited the SEACP region. White catfish (Ameiurus catus) of the family Ictaluridae, which inhabits the freshwater and slightly brackish water drainages along the SEACP and west on the Gulf slope to western Alabama of the USA (Page and Burr, 1991; Schultz, 2003; NatureServe, 2006), might possess the footprints of Quaternary climate change on their mitochondrial DNA. This omnivorous, slow-growing fish is relatively smaller in size with average length 25–35 cm (Schultz, 2003) and attains sexual maturity at 3–4 years. Spawning occurs during spring and early summer when the water temperature reaches approximately 20–21 C. While most of the species within the genus Ameiurus have wide distribution across the drainages of the USA, the A. catus native range is restricted to the SEACP region (Page and Burr, 1991). Fossil based
* Present address: Department of Biology, 208 Mueller Laboratory, The Pennsylvania State University, University Park, PA-16802, USA. E-mail address: [email protected]. 0305-1978/$ – see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2012.02.011
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molecular clocks revealed that A. catus diverged from its common ancestor approximately 16 million years ago, a time that falls within the Miocene era (Hardman and Hardman, 2008; Padhi, 2010). The lack of Miocene-Pliocene fossil evidence of A. catus, however, makes it difficult to predict the geographic range of this species during that time. Nevertheless, the report of late Pleistocene fossil evidence from Vero Beach, Indiana River County, Florida indicates the existence of this species in the SEACP region during that time (Weigel,1963). Although fossil evidence suggests the presence of A. catus in the SEACP region during the Pleistocene, it is difficult to infer the effect of late Quaternary climate change on the population dynamics of the species. The uniparentally inherited, recombination free, and rapidly evolving mitochondrial DNA (mtDNA) has been used widely as a genetic marker to track past population dynamics of vertebrates and invertebrates (Avise, 2000). Using the mtDNA control region (CR) and employing distance-based summary statistics (Fu, 1997), mismatch distribution (Herpending, 1994; Rogers, 1995), parsimony network (Templeton et al., 1995), maximum likelihood (Kuhner et al., 1998) and Bayesian coalescent (Drummond et al., 2005; Drummond and Rambaut, 2007) methodologies, the present study investigates the importance of late Quaternary climate change on the intraspecific diversity and past population dynamics of A. catus.
2. Materials and methods The current distribution and approximate geographic origin of A. catus samples is shown in Fig. 1a. Fin clip tissue from each individual was preserved in 75% ethanol and shipped to the University of Tulsa for genetic analyses. Total DNA was extracted from preserved tissue using the phenol-chloroform method and a portion of mtCR (423 bp) amplified and sequenced using the previously described protocols (Murphy and Collier, 1996). Primers: CR-F: 50 -AACTCTCACCCCTAGCTCCCAAAG-30 and CRR: 50 -CCTGAAGTAGGAACCAGATG-30 (Kocher et al., 1989; Meyer et al., 1990) were used for amplification and sequencing of mtCR. Sequences were aligned using MacClade 4.03 (Maddison and Maddison, 2000). CR haplotypes were identified using Collapse 1.2 (Posada, 2005). Unique haplotypes generated in this study were deposited in GenBank (Accession numbers: EF377366 - EF377387). The appropriate nucleotide substitution model of sequence evolution was selected by the hierarchical likelihood ratio tests (hLRTs) implemented in Modeltest, ver. 3.7 (Posada and Crandall, 1998). A haplotype network was constructed at 95% of connection limit (<9 mutational steps) using TCS 1.18 (Clement et al., 2000). Distance-based mismatch histogram (Rogers and Harpending, 1992; Rogers, 1995; Schneider and Excoffier, 1999), site frequency spectrum based summary statistics such as Fu’s F (Fu, 1997), Bayesian and ML-based coalescent approaches (Kuhner et al., 1998) were utilized to infer the demographic history. Arlequin ver. 3.1 (Schneider et al., 2000) was used to compute the frequency of pairwise differences to evaluate the hypothesis of sudden expansion. Using the same program, Fu’s FS statistic (Fu, 1997) as an indicator of demographic expansion was also estimated. The ML-based growth parameter (g) was estimated using the Markov Chain Monte Carlo (MCMC) method implemented in Fluctuate ver. 1.4 (Kuhner et al., 1998). Ten short MCMCs of 200 generations each and two long chain MCMCs of 20,000 generations were set for each run. The program was run 10 times with different seeds and the mean and standard deviation were estimated from these results of separate runs. Since Fluctuate ver 1.4 may have biased upwards in estimating ‘g’ (Kuhner et al., 1998), a more conservative approach in testing for significance was used (g > 3 g (SD)) (Lessa et al., 2003). Using a rate of 0.015 nucleotide substitutions per site per million year (divergence rate z3% per myr) that was previously estimated for Ictalurus furcatus (Padhi, 2011) as the mean rate, the program BEAST ver 1.48 (Drummond and Rambaut, 2007), which employs a Bayesian MCMC coalescent approach, was used to estimate the time to the most recent common ancestor (TMRCA) and the Bayesian skyline plot (BSP) under a strict clock model with the HKY þ G nucleotide substitution model. The MCMC chains were run for 5 million times to ensure that convergence (allowing a 10% burn-in) was reached. Tracer ver 1.4 was used to check the convergence and to estimate 95% highest posterior density (HPD) of the respective parameters and BSP. The MCMC samples were summarized to infer the maximum clade credibility (MCC) tree using TreeAnnotator v1.4.8 (Drummond and Rambaut, 2007), with posterior probability values shown for the major branches.
3. Results Parsimony network analyses and the MCC tree have revealed the existence of two distinct matrilineal lineages of A. catus (Figs. 1b and 2a). Samples from SEACP formed a unique cluster and are separated from the southeast Gulf coast (SEGC) lineage by five mutational steps (Fig. 1b). Clustering of a few individuals from SEGC with the SEACP clade suggests the possibility of accidental introductions to the SEACP drainage system or the possibility of incomplete lineage sorting. The unimodal pattern of the mismatch distribution of A. catus is best fit to the model of sudden expansion (Fig. 1c). In addition, the significant negative Fs and a significant positive growth parameter (Fig. 1c) further support the hypothesis of sudden expansion of A. catus. Based on the Bayesian coalescent method, SEACP and SEGC lineages are estimated to have diverged from their common ancestor approximately 0.71 (0.37–1.1) million years ago (Fig. 2a) and all the haplotypes appear to have diverged approximately 0.2 myr ago (Fig. 2a), the time that coincides with the late Quaternary epoch. Consistent with the results of mismatch distribution, the BSP has also shown evidence for population expansion. The BSP showed two phase growth: a static phase (phase I) until 0.2 million years ago, followed by a sudden population expansion thereafter (phase II). The timing of haplotype divergence is consistent with the population expansions as revealed by BSP (Fig. 2a and b).
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Fig. 1. Geographic origin of samples, haplotype network, and the mismatch histogram for Ameiurus catus inferred from mtDNA control region sequence data. (a) Map showing geographic origin of samples from Gulf coast and southeast Atlantic coast drainages of USA. 1: Ochlockenee River, Florida (n ¼ 10); 2: Satilla River, Georgia (n ¼ 12); 3: St. Mary River, Florida (n ¼ 11); 4: Savannah River, South Carolina (n ¼ 10), and 5: Roanoke, River, North Carolina (n ¼ 2). (b) Parsimony network analyses showing genealogical relationships among the haplotypes. All the haplotypes were connected at the 95% confident limit. The size of the circle is proportional to the number of individuals. Empty circles are the hypothetical mutational steps. Lines connecting two haplotypes represent one mutational step. Drainages are colour coded. (c) Mismatch histogram showing unimodal distribution is not statistically significant from the model of sudden population expansion. Significant negative Fu’s F and significant positive growth (g) parameter (mean g > 3SD of g) also indicate population expansion.
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Fig. 2. Maximum clade credibility (MCC) tree and Bayesian skyline plot (BSP) showing the timing of haplotype divergence and population dynamics of A. catus. (a) MCC tree shows two distinct lineages representing Gulf coast and SE Atlantic coast. The mean time of divergence (in million years) with 95% highest posterior density (HPD) and the posterior probability (nodal support) for respective nodes are mentioned. Posterior probabilities are in italics. (b) BSP showing two phase growth patterns: a steady growth (phase I) until 0.2 myr followed by sudden growth thereafter (phase II). Genetic diversity measured in terms of effective population size is expressed in logarithmic scale in Y-axis, whereas time in million years (myr) is in X-axis.
4. Discussion Employing distance-based, ML-based, and Bayesian coalescent methodologies and utilizing mitochondrial control region sequence data, the present study reports the importance of the last Ice age on the demographic expansion of white catfish, which is endemic to the southeastern region of the United Sates, the region that never glaciated during the Pleistocene epoch. Although the region never glaciated, the repeated glacial cycles during the last glacial maxima had a profound influence on the drainage patterns of this region (Leigh et al., 2004). Therefore, one might expect that biological entities endemic to this region might possess the footprints of these demographic effects on their mitochondrial DNA. The present study revealed that
98
A. Padhi / Biochemical Systematics and Ecology 42 (2012) 94–98
A. catus is comprised of two distinct matrilineal lineages, SEGC and SEACP, and that their TMRCA is estimated to be 0.7 myr. Results from mismatch distribution, Fu’s F, ML-based coalescent growth parameters, and BSP have also shown evidence of population expansion of A. catus. More importantly, around 0.2 myr all the haplotypes began diverging and the period is marked as a phase of rapid expansion. As observed in the present study for white catfish, previous studies have also reported that several freshwater fishes endemic to the southeastern region of the USA showed genetic discontinuity between Gulf coast and Atlantic coastal plain drainages leading to two distinct matrilineal lineages (Avise, 2000; Soltis et al., 2006). The results of the present study are also consistent with the notion that the late Pleistocene epoch played a dominant role in intraspecific divergence of several aquatic and terrestrial flora and fauna in the southeastern United States (Bermingham and Avise, 1986; Avise, 2000; Soltis et al., 2006). The rapid population expansion and intraspecific diversity of white catfish, which occurred around 0.2 myr, coincides with the time frame when the SEACP region had mostly exhibited braided river patterns (Leigh et al., 2004). The repeated sea level fluctuations during the last ice age (Haq et al., 1987) have had dramatic consequences in changing the dimensions of drainage patterns. Each geographically isolated drainage population with smaller effective population size could eventually result in accumulation of measurable nucleotide differences due to the impact of genetic drift, which is more pronounced for populations with smaller effective population size. The repeated merging and isolation of drainage patterns is therefore believed to have had significant impact on the redistribution of matrilineal lineages (Avise, 2000). Nevertheless, these changing patterns in drainage systems in response to the Quaternary climate change might have had significant impact on the demographic expansion of white catfish and other aquatic species that inhabited the SEACP region. Acknowledgements I thank Dr. Glen Collier for providing laboratory facilities to carry out this work, Dr. Peggy Hill for thoughtful comments towards improving this manuscript and the fisheries biologists of the respective state departments of natural resources for providing samples: Rich Caleteux (Florida), Don Harrison (Georgia), David Allen (South Carolina), and Kent Nelson (North Carolina). I thank Karen Winans for the technical support in DNA sequencing and The University of Tulsa, Office of Research and Sponsored Programs for providing financial support for this study. 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