Extreme genetic divergence in the endemic fish Chirostoma humboldtianum: implications for its conservation

Divergencia genética extrema en el pez endémico Chirostoma humboldtianum: implicaciones para su conservación

García-Martínez Rosa María^{1, 2}, Omar Mejía³, Francisco Javier García-De León⁴ and Irene De Los Angeles Barriga-Sosa²

¹ Doctorado en Ciencias Biológicas y de la Salud, División CBS, Universidad Autónoma Metropolitana Unidad Iztapalapa, Av. San Rafael Atlixco 186 Col. Vicentina, Iztapalapa, D. F. 09340. México.

³ Laboratorio de Variación Biológica y Evolución Departamento de Zoología Escuela Nacional de Ciencias Biológicas-IPN, Miguel Hidalgo, D.F. 11340. México.

⁴ Laboratorio de Genética para la Conservación Centro de Investigaciones Biológicas del Noroeste, S.C. La Paz, B.C.S. 23090, México. email: ibs@xanum.uam.mx

Recibido: 21 octubre 2013.
Aceptado: 29 septiembre 2014.

Abstract

Chirostoma humboltanum is an endemic species widely distributed in isolated basins of Central México. However, habitat alteration had reduced the range of distribution and led to the local extinction of the species in some basins. During the Miocene these basins were connected, allowing dispersal and colonization of new hydrological systems. Later on, tectonic, volcanic and climatic events of the Plio-Pleistocene promoted continuous periods of isolation and reconnection allowing the species evolve through continuous cycles of expansion and contraction of its distribution. Therefore it is expected that these events have left signals in the geographical distribution and genetic diversity and divergence of existing populations. Although the analysis of genetic diversity and genetic structure in the population becomes an important factor for the conservation of a species, few studies have been made in this taxon. In this study we used a 341pb segment of the domain I of the hypervariable region of the mitochondrial control region to analyze the genetic diversity and their distribution in 20 individuals of each one of six lakes located in central México. The values of haplotypic (0-0.938) and nucleotide (0-0.0352) diversity suggested continuous periods of expansion and population contraction related with the formation of the lakes during the Pleistocene which is supported by the BSP and mismatch analysis, and recent anthropogenic factors. In addition, the large number of exclusive haplotypes (66%) and the highly significant genetic differentiation among populations suggests that each one of the population must be conserved because each one is an important component in the evolutionary legacy of the species.

Keywords: Chirostoma humboldtinaum, control region, genetic diversity, population genetics.

Resumen

Chirostoma humboltianum es una especie endémica ampliamente distribuida en cuencas aisladas del Centro de México. Sin embargo, la alteración del hábitat ha reducido drásticamente el área de distribución y llevado a la extinción local de la especie en algunas cuencas. Durante el Mioceno estas cuencas estuvieron conectadas, permitiendo la colonización y dispersión en nuevos sistemas hidrológicos. Posteriormente, los eventos tectónicos, volcánicos y climáticos del Plio-Pleistoceno promovieron continuos periodos de aislamiento y reconexión, permitiendo que la especie evolucionara a través de ciclos de expansión y contracción de su distribución. Por lo tanto, se espera que estos eventos hayan dejado huella en la distribución geográfica y diversidad genética de las poblaciones existentes. Si bien, el análisis de la diversidad genética en una población llega a ser un factor importante para la conservación de una especie, pocos estudios han sido realizados en este taxón. En este trabajo usamos un segmento de 341pb del dominio I hipervariable de la región control mitocondrial, para analizar la diversidad genética y su distribución para 20 individuos de cada uno de los seis lagos localizados en la región central de México. Los valores de diversidad haplotípica (0-0.938) y nucleo-tdica (0-0.0352) sugieren continuos periodos de expansión y contracción poblacional relacionado con la formación de los lagos en el Pleistoceno lo cual es soportado por los análisis BSP y mismatch; y de factores antropogénicos recientes. En adición, la gran cantidad de haplotipos exclusivos (66%) y la alta diferenciación genética significativa entre las poblaciones, sugiere que cada una de las poblaciones debe de ser conservada porque cada una es un componente importante en el legado evolutivo de la especie.

Palabras clave: Chirostoma humboldtinaum, diversidad genética, genética poblacional, región control.

INTRODUCTION

The genus Chirostoma (Pisces: Atherinopsidae) (Saeed et al., 1994; Dyer & Chernoff, 1996) is a group of endemic fishes inhabiting lotic and lentic systems in Central México (Barbour, 1973a; Miller et al., 2005). Although the taxonomic status of the group has not fully resolved, Barbour (1973b) considers that there are 18 species and 6 subspecies. C. humboldtianum (Valenciennes, 1853) is one of the species with the widest geographic distribution. This species is found in geographically isolated lakes and ponds of both clear and turbid water from the valley of Mexico to the Pacific Ocean following the Lerma-Santiago basin system (Miller et al., 2005). Although currently the distribution of the species occurs in isolated basins, lacustrine deposits of the Miocene suggested that the Mesa Central of México was once drained by the vast Lerma-Santiago system (Miller & Smith, 1986) that in turn was connected with other reservoirs that had greater extensions (Barbour, 1973a; Israde-Alcántara, 1997; Moncayo-Estrada et al., 2001). Therefore, it has been pointed that these connections allowed C. humboldtianum populations to disperse and colonize new hydrological systems from the east to the west. The formation of the Mesa Central in the Pliocene, coupled with process of tectonic led to the compartmentalization of the basins, which were intensified by the formation of the Transvolcanic Belt that in turn promoted vicariant events in various aquatic organisms (Webb, 2004; Mulcahy & Mendelson, 2000) and a strong divergence from intraspecific to the interespecific level (Echelle & Echelle, 1984; Barbour, 1973a). Likewise, geological activity, coupled with severe climatic oscillations promoted continuous periods of isolation and reconnections that generated a complex hydrologic system (Moncayo-Estrada et al., 2001; Israde-Alcantara, 1997). The above mentioned events of fragmentation and colonization are expected to leave signatures in the geographical distribution and in the genetic diversity of extant populations. Genetic diversity and geographical distribution often reflects process that occurred during historical time, as well as processes and environmental changes over contemporary time related mainly with the anthropogenic activities (Avise, 2000; Bernatchez & Wilson, 1998; Hewitt, 1996).

In C. humboldtianum, some factors related to human activity such as habitat loss, pollution and overfishing (Alvarez & Navarro, 1957), as well as introduction of non-native fishes (Barbour, 1973a) have recently played important roles in the decline or even disappearance (local extinction) of populations. In spite of this situation documented over almost six decades, the species has not been appointed as threatened in the Official Mexican Standard Norms (NOM-059-Ecol-2001) nor in the IUCN Red List.

The aim in this study is to evaluate genetic diversity and differentiation of C. humboldtianum populations and to elucidate how historical and current factors have influenced its distribution, in order to develop a rational management and conservation program for the species due that the level of genetic diversity reflects the evolutionary potential of a species, which in turn is necessary to enable populations to cope with future environmental changes (Frankham, 2005).

Sampling sites and sequencing of DNA. One hundred and twenty specimens of C. humboldtianum were collected from six locations in Central Mexico (see Fig. 1 & Table 1) and stored in 98% alcohol for mtDNA analysis.

Twenty individuals per locality were used for DNA extraction from ethanol preserved muscle using the salt extraction protocol of Aljanabi & Martínez (1997). A fragment of 360 bp of the hypervariable section I of the mtDNA control region was amplified by PCR using the primers described by Pérez-Ramírez (2005), DloopF (Forward) 5'-GCT CTA ACT CCC AGG AAA ATT-3 ' and DloopR1 (Reverse) 5'-CAC CCC GAT TGC AAC TGT CC-3'. PCR reactions were carried out in a final volume of 25 μl and using a Biorad MyCycler Thermal Cycler (USA). PCR reactions contained 50-100 ng DNA, 1X PCR buffer, 2.5 mM MgCl₂, 2.5 mM dNTP mixture, 10 nmol of "forward" and "reverse" primers and 1U of enzyme Promega GoTaq PCR. The PCR program used includes an initial denaturation step at 96°C for 2 min, followed by 30 cycles of 96°C for 45s, 59°C for 30s, and 72°C for 45s, with a final extension at 72°C for 5min. The PCR products were visualized in agarose gels stained with ethidium bromide. Amplified products were purified using Wizard SV Genomic DNA Purification System Promega kit. The purified PCR products were sequenced in both directions using the PCR primers "forward" and "reverse" in a 10 μl reaction containing 2 μl BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems), 2μl 2X buffer, 1μl of each primer (10 μm), 3μl DNA template (containing approximately 50 ng of DNA) and 2μl of ddH2O water. The thermocycler amplification conditions were: 35 cycles at 96 °C for 30 s, 59.2 °C for 15 s and 60 °C for 4 min. Sequencing reactions were resolved in the automatic sequencer ABI Prism 3100 Avant (Applied Biosystems) at Laboratorio Divisional de Biología Molecular de la UAM-Iztapalapa.

Genetic diversity and population structure. Compiled DNA sequences were edited in BioEdit (Hall, 1999), aligned with Clustal X (Thompson et al., 1997) and checked by eye. Genetic diversity within lakes was determined by assessing the number of haplotypes, polymorphic sites, haplotype (h) and nucleotide diversity (π), using DnaSP ver. 5.0 (Librado & Rozas, 2009).

Population structure was determined using BAPS (Corander et al., 2003; 2004). The Bayesian method implemented in the BAPS software was run under spatial model with the maximal number of groups (K) set from 1 to 20. Each run was replicated six times, and the results were averaged according to the resultant likelihood scores. Additionally the spatial distribution of genetic variation was examined using a hierarchical Analysis of Molecular Variance (AMOVA, Excofier et al., 1992) with significance levels set at α = 0.05 and 10,000 random permutations as implemented in Arlequin 3.5.1.2 (Excoffier & Lischer, 2010). Total genetic variation was partitioned according to the number of clusters defined in BAPS software. On the other hand, genetic differentiation between pairs of population were evaluated a posteriori by means of the pairwise F_ST (Excoffier et al., 1992). The significance of the F_ST value was evaluated by performing a randomization test with 10000 replications with a level of significance of α = 0.05. The diversity indices and F_ST values were calculated using the Tamura-Nei (1993) model with gamma shape distribution parameter of 0.473, which was determined by Modeltest (Posada & Crandall, 1998) as the best evolutionary model for our sequences.

Relationships among populations. The genetic relationships among the resolved haplotypes were reconstructed using the neighbor-joining (NJ) method (Saitou & Nei, 1987) implemented in PAUP (Swofford, 1998). Genetic distances were generated for phylogenetic reconstructions using resolved models of substitution suggested by Modeltest (Posada & Crandall, 1998). The best fit for the control region data was TRN model (Tamura & Nei, 1993) with invariable sites and gamma shape parameters (TRN + I + Γ, I = 0.642, Γ= 0.473, with bases frequencies: A: 0.3582, C: 0.1983, G: 0.1630, T: 0.2806). A bootstrap analysis with 1000 replicates was used to evaluated the support for genetics relationships (Felsenstein, 1985).

In addition, genealogical relationships were examined by means of haplotype networks using the median joining algorithm implemented in Network software (Bandelt et al., 1999).

Demographic patterns. Considering that both historical and contemporary process of basin had contributed to the genetic composition of the species, we tested for possible changes in demographic patterns using several approaches. First, in order to quantify the significant departure from mutation-drift equilibrium, we evaluated the neutrality estimators Tajima's D (Tajima, 1989) and Fu's Fs (Fu, 1997) in Arlequin 3.5.1.2 (Excoffier & Lischer, 2010). Likewise, we performed a mismatch distribution analysis (MMD) of pairwise substitution differences among haplotypes to investigate the demographic history of population by comparing the observed distribution with that expected under the assumption of an expansion model. A unimodal mismatch distribution indicates a recent range expansion; multimodal, including bimodal, mismatch distribution indicates diminishing population size or structured, in contrast a population that has been stationary for a long time become ragged and erratic (Excoffier and Schneider , 1999; Roger and Harpending, 1992; Excoffier et al., 1992), however, the multimodal distributions may also indicate that the population is influenced by migration, is subdivided and/or has undergone a greater number of recent coalescent events (historical contractions) (Ray, et al. 2003; Marjoram & Donnelly, 1994). The smoothness of the observed distribution was quantified by the sum-of-squared deviations and Harpending's raggedness index R (Harpending, 1994) as implemented in Arlequin ver. 3.5.1.2 (Excoffier & Lischer, 2010).

Secondly, we estimated the demographic expansion parameters Γ (MMD age expansion parameter), θ₀ (pre-expansion population size), θ₁ (post-expansion population size), using the generalized non-linear least-square approach. The absolute time of population expansion (t) was calculated through the relationship t = Γ/2u, where Γ measures the time in unit of ½μ generations, μ is the mutation rate per sequence under study per generation and u represents the mutation rate per sequence under study per year (Rogers & Harpending, 1992). We used a time of generation of 1 year and a mutation rate of 3.5% as estimated for the same region in the silverfish Pleuragramma antarcticum (Zane et al., 2006).

As a final step, the demographic history was reconstructed using the Bayesian skyline plot method to detected changes in the effective population size through time using Beast 1.5.1 (Drummond et al., 2005). Four independent Markov chains assuming a strict molecular clock and a coalescent Bayesian skyline model were run for 30 million generations with sampling performed every 10,000 steps. The results of the four independent chains were combined in LogCombiner 1.5.4. (Drummond & Rambaut, 2007) and the Bayesian skyline plots for each population were generated in Tracer 1.5 (Rambaut & Drummond, 2009).

RESULTS

Genetic diversity and population structure. We sequenced and analyzed 341 pb for the control region (CR) in 120 specimens, and recovered 57 polymorphic sites (42 parsimony informative) and 55 haplotypes (Accession KF651987 to KF652041), with almost 66% of unique haplotypes. No insertions or deletions were detected. Overall haplotype diversity was h = 0.938 and nucleotide diversity π = 0.0352 (Table 1). Most haplotypes were defined by substitutions at a single site. Only two haplotypes were shared among individuals from different localities. One of them was also the most common haplotype, found in 27 individuals from the sites Tiacaque (20), Villa Victoria dam (1), Tepuxtepec dam (3), Zacapu lake (1) and San Juanico lake (2), and the other haplotype was found in two individuals from San Juanico lake and San Pedro Lagunillas lake. Seventeen of the haplotypes were shared among individuals within the same localities. The remaining 36 haplotypes (66%) were presented only in single individuals. Haplotype diversity values (h) were high, ranging from 0.789 to 0.979 (Table 1). The exception was the Tiacaque population for which only one haplotype was resolved. Nucleotide diversity (π) analyzed per population showed values ranging from 0.0000 to 0.0384, with the highest value observed in San Juanico (0.0384) and the lowest in Tiacaque (0.00) (Table 1).

BAPS resolved five genetic clusters, with log marginal likelihoods of -1408.6458. The analysis showed a mixture of individuals from different geographic locations in the same clusters. Group A is the most heterogeneous and it is constituted by individuals from Tiacaque dam (20 individuals), Zacapu Lake (20), Tepuxtepec dam (3), San Juanico Lake (5), San Pedro Lagunillas Lake (2) and Villa Victoria dam (1). Group B is conformed by individuals from Villa Victoria dam (19), San Juanico Lake (5) and Tepuxtepec dam (1). Group C include individuals from Tepuxtepec dam (16), San Juanico Lake (5) and San Pedro Lagunillas Lake (1). Group D is formed of individuals from San Pedro Lagunillas Lake (17) and group E was constituted with the specimens from San Juanico Lake (5).

An Analysis of Molecular Variance (AMOVA) revealed that 70.30% of the total variation corresponds to variation unshared among BAPS defined groups, 10.15% is variation unshared among population within a group and 19.55% is variation distributed within populations. The fixation indices shown significant differences in the three levels after 10000 randomizations (F_CT = 0.030, P < 0.001; F_SC = 0.3418, P < 0.001; F_ST = 0.8045, P < 0.001) (Table 2).

The F_ST paired values were high and significant, and ranged from 0.1712 for San Juanico lake-Tepuxtepec dam up to 0.8288 for Villa Victoria dam-Tiacaque (Table 3).

Relationships among populations. The generated NJ of the 55 resolved haplotypes allowed the identification of the same five groups recovered by BAPS, and analyzed in AMOVA, with bootstrap support higher than 60 % (Fig. 2).

The median joining network exhibits a pattern consistent with a complex demographic history and little geographic structure (Fig. 3). Haplotype A01 is widely distributed, occurring at high frequency in Tiacaque dam and also being present in four of the five basins (Villa Victoria dam, Tepuxtepec dam, San Juanico and Zacapu lakes). Haplotype A10 was shared between two individuals from different localities (San Juanico and San Pedro Lagunillas), 17 of the 55 haplotypes were shared by individuals of the same location and the remaining 36 haplotypes were found each at a single individual. The five groups recovered in the Bayesian analysis and NJ was also resolved in the haplotypes network.

Haplogroups A and B are separated by seven mutational steps, and those groups are separated from C, D and E by nine nucleotide substitutions (Fig. 3). Groups C and D are separated each one only by four mutational steps, while group C and E are separated by ten nucleotide substitutions. Haplotypes within groups were mostly separated by one or two nucleotide substitutions; however, groups C and D, shows some haplotypes separated by six substitutions.

Demographic patterns. The histograms of the mismatch distribution (MMD) of the entire population showed a bimodal distribution that deviated from the expected distribution under the sudden expansion model (Fig. 4). This outcome is supported by lack of significance of the Tajima's D and Fu's Fs tests. On the other hand, the MMD in Group A showed L-shaped distribution, which represent a balanced distribution. Groups B, C and D showed an unimodal distribution that suggests an expansion process (Fig. 4). The MMD for group E was not performed because this group is only comprised by five individuals and demographic analyzes lose their validity under a very low sample size. In all cases, raggedness indices (r) were not significant, thus not allowing us to reject the null hypothesis of stationarity (Table 4). The examined populations displayed negative non-significant Tajima's D values. Similarly, the calculated Fu's Fs values were negative, and only group D showed a significant value with a time since expansion of 225,178 years ago (τ= 2.6875) (Table 4). We estimated that during the expansion, the population increased from θ₀ (1.4641) to θ₁ (225). The Bayesian Skyline Plot analysis for group D also indicates a population expansion during the last 300,000 years. Group A, B show a stable trend in population size in the last 200,000 years, and C resolved also as stable since the last 250,000 years (Fig. 5). There is not graph for group E for the reasons previously mentioned.

DISCUSSION

Ward et al. (1994) suggested that freshwater fish in general terms exhibit lower levels of genetic diversity in comparison to marine fish using allozyme markers. Other works using allozymes markers had shown low genetic diversity levels for this and other species of the genus Chirostoma, although such results might be related to the low polymorphism exhibited by those markers (Barriga-Sosa et al., 2004) and to the limited number of specimens analyzed (6-8 individuals) (Barriga-Sosa et al., 2002). In contrast to the result reported by Ward et al. (1994), we recovered a relatively high number of haplotypes (55) with a mitochondrial marker in 120 analyzed specimens, with almost 66% of unique haplotypes, and high average values of both haplotype and nucleotide diversity (h = 0.938 and π = 0.035). For other species of freshwater fishes, lower values of genetic diversity have been reported using the same mitochondrial region. For instance, in fish living in cenotes, Vázquez-Domínguez et al. (2009) resolved 5 haplotypes in 56 individual and average haplotype and nucleotide diversity of 0.15 and 0.001 respectively; in the atherinomorphs, Atherinomorus endrachtensis (Quoy & Gaimard, 1825), Gotoh et al. (2011) found 72 haplotypes in 205 individuals, h = 0.151-0.593 and n = 0.00026-0.0029, whereas for the close relative Odontestes argentinensis (Valenciennes, 1835), Beherengaray & Sunnucks (2001) reported what they considered high values of h = 0.85-0.94 and π = 0.014-0.018.

The analysis of genetic diversity in the examined population of C. humboldtianum exhibited greatly variable values. Grant & Bowen (1998) have proposed that high values of nucleotide diversity (π) and haplotype diversity (h) found in San Juanico lake, Tepuxtepec dam, Villa Victoria dam and San Pedro Lagunillas lake and Zacapu lake (Table 1), are characteristic of a stable population with long evolutionary history or population admixture of differentiated lineages. However, the genealogical analysis does not support a long evolutionary history for the species, as can be observed by the absence of deep branching among haplotypes (Fig. 2). This result indicates that C. humboldtianum is a recent species (Moncayo-Estrada et al., 2001, Echelle & Echelle, 1984) that diversified ca. 0.52 mya (Bloom et al., 2013). Furthermore, we believe that the admixture hypothesis is more likely for the species, since each geographic population consists of a mixture of haplotypes that do not show geographic pattern (Fig. 1).

The complex geological history of the Mesa Central in the Plio-Pleistocene caused several periods of isolation and reconnection among basins (Domínguez-Domínguez et al., 2008; Miller et al., 2005; Moncayo-Estrada et al., 2001; Israde-Alcántara & Garduño-Monroy, 1999; Barbour, 1973a; Tamayo & West, 1964). In first instance, the separation of basins produced geographic isolation. This separation is well documented for the late Pleistocene and early Holocene (Alvarez & Moncayo, 1976; Barbour, 1973a). This geographic isolation is reflected in the high and significant genetic differentiation found among basins/groups in this study. On the other hand, the periods of reconnection favored gene flow among previously isolated populations. Additionally, climatic oscillations related to Pleistocene glaciations caused many cycles of contraction of geographic ranges during cold periods and expansion during subsequent warming (Bradbury, 2000; Israde-Alcántara & Garduño-Monroy, 1999; Ferrari et al., 1999). In this sense, both analyses the mismatch distribution and Bayesian Skyline plot support the hypotesis of a rapid population growth of C. humboldtianum during this period. Likewise, the global MMD suggest connections and gene flow among the basins in the past or multiple events of population expansion.

The hypothesis of sudden expansion of group D is also supported by the negative Fu's Fs values and the star-shaped haplotype network (Fig. 3). This type of network is characteristic in populations that have experienced a recent population expansion from a smaller number of founders (Avise, 2000). The Bayesian skyline plot showed that the group D experienced an expansion process 200,000 ~ 300,000 years ago (Fig. 5); furthermore the expansion was estimated to be ~ 225,178 years ago from the sudden expansion model (τ = 2.26875) (Table 4). This estimation suggests that the expansion occurred in the middle Pleistocene (between 126,000 and 781,000) when volcanic and tectonic activity were more intense in the Mesa Central causing the overflow of the Chapala basin towards the ancestral Río Grande de Santiago and conducting to the isolation of Río Lerma and the formation of modern Ameca and Lerma-Santiago rivers (Miller et al., 2005, Barbour, 1973a).

Our results of MMD suggest that the expansion time of the group C and group B was during middle Pleistocene (594,855 and 458,215 years ago, respectively) (τ = 2.6875) (Table 4). Previous studies and historical inferences based on the fish fauna of this region have reported a series of paleolakes that joined the Valle de México and Río Lerma (Domínguez-Domínguez et al., 2008; Moncayo-Estrada et al. 2001; Israde-Alcántara & Garduño-Monroy, 1999; Barbour, 1973a; Tamayo & West, 1964). However, during the Plio-Pleistocene these connections were broken by tectonic and volcanic activity (Doadrio & Domínguez, 2004; Israde-Alcántara & Garduño-Monroy, 1999). During late Pleistocene to early Holocene a successive reconnection between the Valle de México and Río Lerma led to a new contact between species and to a genetic exchange through NE-SW and E-W faulting, which cuts the Sierra de las Cruces (Israde- Alcantara & Garduño-Monroy, 1999; Barbour, 1973a). The above mentioned is supported by the fossil record of fishes of the Valle de México, which currently are not distributed in this basin (Alvarez & Moncayo, 1976).

The complex recent geological processes of Central México region were decisive in shaping the genetic structure of this species, however, complexity increased by human activity. For instance, it is documented that during the 70's translocations of species of "peces blancos" and minnows were conduced into several water reservoirs of México including Michoacán and Estado de México (Rosas, 1976). In this regard, Alvarez (1963) and Barbour (1973a) referred to Chirostoma consosium consocium and C. c. reseratum as native species of San Juanico lake, however, E. Soto-Galera (per.com. Laboratorio de Ictiología y Limnología) reports the current presence of C. humboldtianum in the basin. Furthermore and although the presence of the species in Zacapu Lake is referred by several authors as part of its original distribution (Paulo-Maya et al., 2000; Barbour, 1973a; Alvarez, 1963), Medina Nava (1997) mentions its introduction in this basin, however, she did not mention possible year(s) of introduction. The species has also been reported in lakes Chapala, Pátzcuaro and Zirahuén (Barriga-Sosa et al., 2002; Alaye, 1993; Alvarez & Navarro, 1957; Barbour, 1973b).

The present study allows concluding that the genetic structure of C. humboldtianum can relate to geological and climatic events of the Plio-Pleistoceno, although we cannot discard that translocations could explain the presence of the shared haplotypes in different geographical location. Thus, our results prompts for futher genetic population studies designed to determine the level of integrity and/or genetic introgression of the species and closely related species along its range of distribution.

Conservation implications. México has 375 freshwater fish species, 60% native (Miller, 1986). Anthropogenic factors such as habitats destruction, introduction of exotic species, pollution and eutrophication threaten wildlife inhabiting freshwater reservoirs (Miller et al. 2005).

The results of the present study might contribute to the development of conservation strategy plans for C. humboldtianum, as has been previously proposed by Barriga-Sosa et al. (2002), whom reported a strong genetic population differentiation in the species as it is also resolved in the present study (see pairwise F_STvalues).

Although we are aware of the possible evolutionary stochastic effects and of the limitation of defining conservation units only on mtD-NA results, we propose that five of the six analyzed populations could be proposed as candidates to be preserved under legal protection, since all of them contain a portion of the total variation of the species. Each of these geographic locations can be tentatively defined as an ESU (Evolutionary Significance Unit), since each ones is a segment of the population or group of stocks that are reproductive isolated from other conspecific, or unrelated population and represent an important component of the evolutionary legacy of the species (Waples, 1995). However, further studies using other molecular markers as microsatellites or SNPs, can aid in corroborating this proposal.

ACKNOWLEDGEMENTS

This study is part of the PhD thesis of the first author at Doctorado en Ciencias Biológicas y de la Salud at Universidad Autónoma Metropolitana-Iztapalapa. Fellowship CONACYT-224707 was granted to Rosa María García Martínez. Financial support was received from Consejo Nacional de Ciencia y Tecnología (CONACYT-CB-2009-01-130220) and UAM.147.07.03/147.09.0. Mexican government kindly issued permit number DGOPA.07343.310810.4128 to conduct this research. We thank the Biologist Ramón Cisneros, M. en B. Laura Georgina Núñez García and the fishermen of each collection site for support in the capture and maintenance of organisms. We also thank Dr. Joel Paulo Maya for their help in specimens identification.

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