Elena G. Gonzalez1 1,2 , Mar Torralva , and Ignacio ...digital.csic.es/bitstream/10261/197601/1/679698.pdf · Ciencias Naturales, MNCN-CSIC, José Gutiérrez Abascal, 2, Madrid, Spain,

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Phylogeography and population genetic analyses in the Iberian toothcarp

(Aphanius iberus Valenciennes, 1846) at different time scales

Elena G. Gonzalez1, Carina Cunha1, Hamid R. Ghanavi1,2, Francisco J. Oliva-

Paterna3, Mar Torralva3, and Ignacio Doadrio1

1. From the Departamento de Biodiversidad y Biología Evolutiva, Museo Nacional de

Ciencias Naturales, MNCN-CSIC, José Gutiérrez Abascal, 2, Madrid, Spain, 28006;

2. Biology Department Sölvegatan 37, 22362 Lund, Sweden and the

3. Departamento de Zoología y Antropología Física, Universidad de Murcia; 30100

Murcia, Spain

Address correspondence to E. G. Gonzalez at the address above, or e-mail:

[email protected]

Downloaded from https://academic.oup.com/jhered/article-abstract/doi/10.1093/jhered/esx076/4259313/Phylogeography-and-population-genetic-analyses-inby Museo Nacional de Ciencias Naturales useron 04 October 2017

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Abstract

Secondary freshwater fish species inhabiting fluctuating and extreme environments

are susceptible to changes in dispersion, effective population size, and genetic

structure. The Iberian toothcarp Aphanius iberus is an endemic cyprinodontid of the

Iberian Peninsula restricted to brackish water of salt marshes and coastal lagoons on

the eastern Spanish Mediterranean coast. In this study we analysed mitochondrial

cytochrome b DNA and microsatellite variation to evaluate ways in which the

processes of extinction, dispersal, and colonization of A. iberus across its geographic

distribution has affected its population genetic structure over time and space. The

Aphanius iberus network reconstruction indicated subtle levels of phylogeographic

structuring. This, combined with substantial mtDNA genetic diversity, suggest that

Pleistocene glaciations had a lesser effect on the demographic structure of its

populations than was the case for Iberian fresh-water species with similar

distribution. Haplotype network, hierarchical AMOVA, and pairwise st comparisons

involving some Levantine samples showed a relatively high degree of mtDNA

differentiation, which could be explained by historical isolation of the Villena Lagoon

population. Conversely, significant genetic differentiation, following an isolation-by-

distance pattern, and a reduction in Ne though time was detected with

microsatellites, suggesting extensive habitat fragmentation on the Mediterranean

coast of the Iberian Peninsula over the past hundreds of years. At a smaller

geographical scale (Mar Menor Lagoon), habitat fragmentation, probably due to

human activity, appears to have resulted in substantially reduced migration and

increased genetic drift, as shown by expanded genetic differentiation of populations.


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Subject areas: conservation, Cyprinodotidae, fragmented populations, microsatellite,

mtDNA, secondary freshwater fishes,


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Introduction

Fluctuating environmental conditions can influence species dispersal capacity and

population structure (Schönhuth, Luikart and Doadrio, 2003; Whitehead, 2009).

Assessment of gene flow is important for conservation of species that are susceptible

to temporal bottlenecks and local extinctions due to seasonal flooding and drought

events and the consequent changes in effective population size. Low or reduced

periods of gene flow, as seen with habitat fragmentation, can lead to local inbreeding

depression, whereas high or increased periods of gene flow can produce

physiological and behavioural changes in response to environmental changes (Reed

et al., 2011; Watling and Donnelly, 2006). Periods of disturbance over time can affect

the probability of extinction-colonization processes and the survival of migrants;

hence habitat connectivity greatly mediates evolution and species persistence

(Lafferty, Swift and Ambrose, 1999; Watts et al., 2015).

Cyprinodontids are short-lived fishes comprising several species inhabiting

brackish or fresh waters and well adapted to fluctuating and extreme environmental

conditions, including exhibiting high tolerance for hypersaline waters and elevated

water temperatures (Leonardos, Sinis and Petridis, 1996; Martin and Saiki, 2005;

Oltra and Todolí, 2000). The Iberian toothcarp Aphanius iberus (Valenciennes 1846)

is an endemic cyprinodontid of the Iberian Peninsula, restricted to brackish water of

salt marshes, coastal lagoons, and river mouths on the eastern Spanish

Mediterranean coast (Oliva-Paterna, Torralva and Fernández-Delgado, 2006).

Anthropogenic habitat degradation and fragmentation has resulted in a decline in


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populations (Araguas et al., 2007; Casas et al., 2011), which currently show a more

fragmented distribution compared to historically (Oliva-Paterna and Torralva, 2008)

(Figure 1). The species has been recorded in the Catalonian, Levantine, and Murcian

(sensu) biogeographic areas (Doadrio, Perdices and Machordom, 1996). Alterations

to their natural habitat have occurred over an extended period of time, to the point

that there are currently no areas that have not suffered anthropogenic pressure,

mainly agricultural and urban. A paradigmatic case is the endorrheic Villena Lagoon

(Alicante, Spain), the only inland lagoon in which A. iberus is known to occur. The

lagoon was drained in 1803, and the land was converted to farmland, with the water

diverted to the Vinalopó River by a drainage canal. Small populations of A. iberus

were discovered near the original Villena Lagoon, one in 1992 in the canal and the

other in 1999 in a nearby irrigation pond. The entire populations were collected and

maintained in a fish hatchery to prevent their loss in case of disappearance of the

water bodies in which they were found. Restriction fragment length polymorphism

analysis of mtDNA markers (Fernández-Pedrosa et al., 1995), cytochrome b

sequences (Perdices et al., 2001), and morphological data (Doadrio, Carmona and

Fernández-Delgado, 2002) identified the Villena Lagoon population as divergent from

two other Mediterranean coast A. iberus populations. To further characterize unique

aspects of the Villena population, it is necessary to increase the number of

populations sampled and the types of markers analysed (such as nuclear DNA).

Aphanius iberus was historically found in coastal habitats, including the Mar

Menor Lagoon (Murcia, Spain) (Figure 1). This lagoon comprises shallow littoral

areas and several adjacent wetlands that represent examples of an extreme

heterogeneous environment, especially with respect to salinity (Pérez-Ruzafa et al.,


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2005), sustaining a diverse fish community (Oliva-Paterna et al., 2006; Verdiell-

Cubedo et al., 2008). It also presents a good illustration of recent human disturbance

and artificial fragmentation. The lagoon is separated from the Mediterranean Sea by

a sand bar ca 22 km in length that allows the exchange of water from the sea through

two shallow channels, El Estacio and Las Encañizadas (Figure 1). El Estacio was

dredged in the early 1970s to make it navigable, altering the water recycling periods

with the Mediterranean Sea. The average salinity ranges from 39 to 46 ppt, higher

than the Mediterranean Sea, due to evaporation and the restricted exchange (Pérez-

Ruzafa et al., 2005). The lagoon is also influenced by terrestrial watercourses, such

as the seasonal and torrential rainfall rivers typical of the Mediterranean climate, that

create sudden environmental shifts. Although A. iberus is a resident species of the

shallow littoral areas in the Mar Menor coastal lagoon (Oliva-Paterna et al., 2006),

little is known about the population biological traits and genetic structure in these

areas, and only reports of its presence and relative densities have been published

(Oliva-Paterna and Torralva, 2008).

In addition to human pressure, distribution has been recently subjected to

predation by, and competition with, invasive species (Doadrio, 2011; Doadrio et al.,

2002; Gutiérrez-Estrada et al., 1998) displacing A. iberus from fresh and oligosaline

waters to more saline waters, primarily eusaline and hypersaline (Alcaraz, Bisazza

and García-Berthou, 2008; Caiola and De Sostoa, 2005; Rincón et al., 2002). Given

the effects of these human-mediated and natural and artificial ecological events

(Oliva‐Paterna et al., 2009; Verdiell-Cubedo et al., 2014), dramatic differences in the

genetic pattern across the geographic range of A. iberus would be expected. Life

history traits such as rapid growth, early maturity, and a generation time of


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approximately two per year, (García-Berthou and Moreno-Amich, 1992; Oltra and

Todolí, 2000) could have counteracted the impact of population declines on genetic

variation (Schönhuth et al., 2003). However, little is known about the extinction-

colonization dynamics of the species or how the processes of extinction, dispersal,

and colonization are affecting its population genetic structure across time and space.

Habitat fragmentation or environmental shifts could be the source of the genetic

structure previously detected based on otoliths, allozymes, and mitochondrial DNA

(Araguas et al., 2007; Doadrio et al., 1996; Fernández-Pedrosa et al., 1995; Perdices

et al., 2001; Reichenbacher and Sienknecht, 2001), and the lower nuclear genetic

variation observed when compared with other Aphanius species (Gonzalez, Pedraza-

Lara and Doadrio, 2014; Pappalardo et al., 2015).

Here, we analyse genetic data from the mitochondrial cytochrome b gene and

microsatellite nuclear markers in 439 specimens of A. iberus to estimate the level

and distribution of genetic variation. Effective migration rates among 20 populations

were examined to assess the relative influence of migration and genetic drift on

genetic variation at historical and contemporary time scales. Bayesian clustering, as

well as classical genetic-variance-based methods (hierarchical AMOVA test, Fst

pairwise comparisons and isolation-by-distance tests), were used to study population

genetic differentiation.


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Materials and Methods

Genetic sample collection and DNA extraction

A total of 439 A. iberus specimens were collected from 20 sites along the

Mediterranean coast of the Iberian Peninsula, covering most of the species

distribution (Figure 1, Table 1). Of these, 236 specimens were included in a more

exhaustive survey conducted at the Mar Menor Lagoon, Murcia. Four specimens

(two from AE and another two from PA) were processed only for the mtDNA dataset.

Specimens corresponding to Albuixec (Alb), Sax (Sax), and Villena (Vil) (Table 1)

were obtained from captive populations used in breeding programmes at the

Protection and Study of Natural Environment Center (Conselleria del Medi Ambient,

Comunidad Valenciana, Spain). The original Albuixec area was a marsh near

Valencia that was drained in 1991. Prior to draining, most A. Iberus and Valencia

hispánica (another endemic cyprinodontid of the Iberian Peninsula) were captured

and moved to the breeding facilities at Valencia. The populations of Sax and Villena

were collected near the original Villena Lagoon. The individuals from Albuixec

belonged to the third generation of breeding stock, whereas those from Sax and

Villena were collected in the wild and subsequently transported to the breeding

facilities. Fish from the wild populations were captured using a dip-net under local

authority permission, and a small fragment of the caudal fin was taken before the fish

was released at the same point in which it was caught. For that reason, we consider

both captive and wild specimens included in the study as from the original natural

stock.


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Tissue was preserved in 95% ethanol and stored at -20 ºC. Genomic DNA was

extracted using the Qiagen DNeasy tissue kit (Qiagen) following manufacturer’s

instructions. Voucher samples were stored in the fish collection and at the DNA

collection of the Museo Nacional de Ciencias Naturales of Madrid (MNCN-CSIC),

Spain.

Mitochondrial DNA analyses: DNA amplification and genetic diversity

The complete mtDNA cytochrome b (cytb) gene from 173 specimens of A. iberus

was amplified according to PCR conditions described in Gonzalez et al. (Gonzalez et

al., 2014). Fragments were sequenced on an ABI 3730XL DNA sequencer.

Alignment of the nucleotide sequences was performed using the default pairwise and

multiple alignment parameters in CLUSTAL X 1.83, implemented in MEGA 6.0

(Tamura et al., 2013) and verified manually to maximize position homology.

The cytb mtDNA sequences were analysed with DnaSP 5.10 (Librado and

Rozas, 2009) to determine the number of haplotypes (Nh), mitochondrial haplotype

diversity (Hd) (Nei, 1987), and nucleotide diversity (π) (Nei, 1987) with their standard

deviations (SD). The Catalonian, Levantine, and Murcian Operational Conservation

Units described by Doadrio et al. (Doadrio et al., 1996) were used as criteria for

dataset analyses. To assess the phylogeographic history of A. iberus along its

distribution, we reconstructed a network for cytb sequences using HAPLOVIEW

(Salzburger, Ewing and Von Haeseler, 2011).

Mitochondrial DNA analyses: demographic and population structure


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To evaluate the influence of seasonal fluctuations on historic demography of A.

iberus, deviations from a model of mutation-drift equilibrium were tested using

Tajima’s D (Tajima, 1989) neutrality test, Ramos-Onsins and Rozas’ R2 (Ramos-

Onsins and Rozas, 2002), and Fu’s Fs (Fu, 1997) tests implemented in DnaSP 5.10,

and their significance was assessed using 1000 coalescent simulated re-samplings.

For neutral markers and under a population expansion model in populations that

have experienced recent expansion, significant low and negative values would be

expected.

The Φ-statistics (Excoffier, Smouse, and Quattro, 1992) were estimated using

ARLEQUIN v. 3.5 (Excoffier and Lischer, 2010), and their significance was determined

with 104 random permutation tests. The hierarchical distribution of mitochondrial

genetic variation among populations was determined using an analysis of molecular

variance (AMOVA) as implemented in ARLEQUIN 3.5 (Excoffier and Lischer, 2010).

We examined the overall differences of the complete dataset (one gene pool) and on

population groups based on an a priori geographic division of sampling locations.

Finally, the Mar Menor Lagoon populations were analysed separately. In all instances

with multiple tests, P-values were adjusted using sequential Bonferroni correction

(Rice, 1989).

Microsatellite DNA analyses: loci amplification and genotyping

To test the validity of microsatellite cross-species amplification, eight microsatellite

loci characterized for Aphanius fasciatus (Babbucci et al., 2007) and 11 loci


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described for Cyprinodon (Strecker, 2006) were analysed in a subsample of 25 A.

iberus. The PCR conditions used are provided in Gonzalez et al. 2014. Sixteen of the

19 loci tested produced a reliable PCR product, and each forward primer was

labelled with fluorescent dyes (Invitrogen). Amplified PCR products were run on an

ABI Prism 3730 DNA Analyzer (250-500 LIZ size standard). Allele scoring was

conducted using GENEMAPPER 3.7 (Applied Biosystems). Of these loci, seven were

polymorphic and were used in further analyses (see Table S2). A total of 435

samples were amplified following the described protocol. Approximately 5% of the

samples were re-amplified to ensure scoring repeatability.

Microsatellite DNA analyses: genetic diversity

Genetic diversity was calculated for each sampling site and locus. Exact tests for

departure from Hardy–Weinberg equilibrium were conducted in GENEPOP 4.2.1

(Rousset, 2008), and linkage disequilibrium was mapped using the Markov chain

algorithm, following dememorization of 10 000 steps with 20 batches and 5000

iterations per batch (Guo and Thompson, 1992). The site-specific average inbreeding

coefficient (FIS) and allelic richness (RS) were calculated using FSTAT (Goudet, 2001).

The average number of alleles across all loci (Na) and observed and expected

heterozygosity (HO and HE, respectively) were obtained using GENETIX 4.05 (Belkhir

et al., 2004). Unbiased HE (Nei, 1987) was used due to the small sample size of

some populations. In all cases, the p-values were adjusted with Bonferroni

procedures to correct for the effect of multiple tests (Rice, 1989).


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A bimodal test for each locus and sampling site was performed to detect

possible genotyping errors resulting from preferential amplification of one of the two

alleles, misreading of bands, or transcription errors, using the program DROPOUT

(McKelvey and Schwartz, 2005). We also tested for the presence of null alleles and

scoring problems associated with allelic stuttering or allelic dropout using MICRO-

CHECKER 2.2.3 (Van Oosterhout et al., 2004). These analyses did not show

evidence of stutter bands or genotyping errors, and null allele frequency was low.

Since adjusting frequencies to take into account null alleles (Brookfield, 1996) did not

affect inbreeding coefficient estimates, all loci were used in further analyses.

Microsatellite DNA analyses: population differentiation

Genetic differentiation among populations was assessed based on the FST statistic

(Weir and Cockerham, 1984). Significance level was assessed by conducting 10 000

permutations in ARLEQUIN 3.5 (Excoffier and Lischer, 2010). As with the mtDNA

cytb data, AMOVA was used to assess the partitioning of microsatellite variation in

allelic frequencies (FST) between and within grouping schemes of geographic

differentiation.

We also tested the hypothesis of isolation-by-distance across sampling sites

using a linear stepping-stone model of migration (Rousset, 1997), under the null

hypothesis of no correlation between pairwise estimates of corrected (logarithm) and

uncorrected FST vs. geographic distances. Mantel tests (Mantel, 1967) were applied

in IBD 1.52 (Bohonak, 2002) with 10 000 randomizations. Again, this analysis was

performed separately for all data and the Mar Menor Lagoon samples.


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Finally, the patterns of population structure were analysed using the model-

based Bayesian clustering procedure in STRUCTURE 2.3.4 (Falush, Stephens and

Pritchard, 2003), which assigns individuals to K populations based on their multilocus

genotype. In STRUCTURE we assumed an admixed model and a uniform prior

probability of the number of populations, K. The modal value of lambda, ∆K (Evanno

et al. 2005), was also calculated to infer the best value of K. Five replicates for each

run were performed for K = 1-25, and MCMC consisted of 105 burn-in iterations

followed by 106 sampled iterations.

Microsatellite DNA analyses: gene flow and population bottleneck detection

Two methods were used to determine levels of gene flow for A. iberus populations at

different time scales, and the results were compared. Contemporary gene flow

among A. iberus populations was determined with the software BAYESASS+ 1.3

(Wilson and Rannala, 2003), which estimates the migration rates (m) based on the

inferred proportion of immigrants within the past 2-4 generations. Samples were run

for 3 x 107 generations with a burn-in of 106 generations. Samples were taken every

2000 generations. Two independent runs were performed to check convergence of

results.

The software MIGRATE 3.2.6 (Beerli, 2009) was used to estimate historical gene

flow, the effective population size, Ne, and the number of migrants per generation,

Nm. The method is based on a coalescence model with mutation-scaled migration

rates, M (M = m/μ, where m is the migration rate per generation among populations

and μ denotes mutation rate). We assumed a full model and symmetrical rates. An


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MCMC run consisted of ten short chains sampling 50 000 trees, three long chains

sampling 500 000 trees, and an adaptive heating scheme. MIGRATE also estimates

the mutation-scaled effective population size , defined as , where Ne is the

effective population size. Ne, was calculated using a mutation rate of 10-3 as

assumed for nuclear-encoded loci with tetra- and di-nucleotide repeats in pink

salmon Oncorhynchus gorbuscha (Steinberg et al., 2002). Moreover, the number of

migrants per generation, Nm (product of and M divided by four) was also

calculated.

To compare migration rates generated by BAYESASS+ and MIGRATE, the

values of m directly obtained with BAYESASS+ were plotted against the values of m

estimated by MIGRATE (using a mutation rate of 10-3, (Steinberg et al., 2002)). A

paired t-test was conducted on pairwise values to assess whether estimates were

significantly different.

Possible severe reductions in effective population size were assessed using

BOTTLENECK (Dirienzo et al. 1994; Piry et al. 1999). Analyses were carried out

assuming three mutation models, (i) an infinite allele (IAM), (ii) a stepwise mutation

(SMM), and (iii) a two-phase (TPM) with 70% stepwise and 30% variable, applying

the Wilcoxon signed rank test for statistical detection of He excess. Estimation was

based on 10 000 replicates. The mode-shift test (Luikart et al. 1998) in

BOTTLENECK was conducted to determine whether the observed distribution of

allele frequencies among A. iberus populations differed from that expected under

drift-mutation equilibrium.


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Results

Intra-population mtDNA genetic diversity

Sequences for the complete cytb gene were trimmed to the size of the smallest

fragment, and alignments produced a dataset of 1,117 base pairs (bp) (GenBank

accession numbers in Table S1). Polymorphic sites identified 59 haplotypes among

the 173 fish analysed for the mitochondrial cytb gene, including 13 unique

haplotypes. Ninety-three sites were polymorphic, 44 of which were singleton variable

sites, and 49 were parsimony-informative. The mtDNA sequence variation was

characterised by global nucleotide and haplotype diversities of π = 0.0051 and Hd =

0.848, respectively. Haplotype and nucleotide diversity of the Levantine populations

were higher than those of Murcian populations, while the Mar Menor Lagoon

populations showed the lowest genetic diversity (Table 2).

The haplotype network showed a subtle geographic structure among

mitochondrial haplotypes and indicated the existence of two main haplogroups in a

star-like topology, pointing to rapid population expansion over a short time (Figure 2).

The majority of haplogroups showed a low number of substitutions differentiating

haplotypes, which were commonly shared among locations and did not fully

correspond to the geographic distribution of the samples. Within the most common

haplogroup, one subgroup comprised individuals from Mar Menor Lagoon (Rib, SPe,

Enc, Cie, Mch, PA, PLV, Urr, Cam, and Hit), Ramblas de las Moreras (RMo), and

Pinillas (RPi); while the other subgroup was represented by the remaining A. iberus

populations, together with a few individuals from RMo and Rib. In addition to the


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main haplogroup, we found a subgroup of diverged haplotypes comprising individuals

from Villena (Vil) and Sax (Sax), separated from the rest by 15 mutational steps, and

a subgroup comprising the individuals from the Catalonian region (AE), separated by

five mutational steps. The main diverged subgroup corresponded to the captive

specimens taken from the Villena Lagoon.

Mitochondrial historical demography and genetic structure

The neutrality test gave significant results with Tajima’s D (Tajima, 1989), Ramos-

Onsins and Rozas’ R2 (Ramos-Onsins and Rozas 2006), and Fu’s Fs (Fu, 1997)

tests for all samples; hence a sudden demographic expansion model cannot be

rejected (Table 2). Analyses of different geographic groupings produced mostly

negative D and Fs values, indicating an excess of rare variants, as would be

expected from a past population expansion.

Pairwise ΦST values are presented in Table S3. The highest differentiation was

found among the Sax (Sax), Vinalopó (Vin), and Adra (Adr) populations; whereas the

lowest pairwise values were seen in Mar Menor Lagoon populations. The hierarchical

analysis of molecular variance (AMOVA) strongly supported high and significant

genetic differentiation among and within A. iberus populations (Table S4). Results

indicated overall significant genetic structuring (P <0.0001) of the analysed samples

(overall FST = 0.63). The genetic variability was primarily explained by variation

among populations within regions. Similarly, genetic structuring was observed among

regions when samples were grouped according to region.


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Intra-population nuclear genetic diversity

All loci were polymorphic in each population, with the mean number of alleles (NA)

per sample (in all loci) similar among populations, varying from 2.7 alleles in Rambla

de las Pinillas (Rpi) to 5.7 alleles in Marchamalo (Mch) (mean NA ± SD, 4.1 ± 0.9).

The allelic richness also showed similar values among samples, with a mean of

3.81± 0.43 alleles (Table 3). The mean number of private alleles (PA) was 0.9 per

population, ranging from 0 to 4 (Table 3). The average values of nuclear diversity,

measured as NA and HO, of the captive populations from Sax (Sax), Villena (Vil), and

Albuixec (Alb) was not significantly different from the wild populations (p > 0.05). No

evidence of linkage disequilibrium was found, as none of the corresponding exact

tests remained significant after Bonferroni correction; thus, microsatellite loci were

considered statistically independent. Overall, the samples from Mar Menor Lagoon

showed the highest levels of genetic diversity as measured by HE and Rs, while the

Adra (Adr) population possessed the highest number in private alleles. The mean HO

for all populations and in all loci (0.52 and 0.39, respectively) was slightly high.

However, most of the population showed deviation of Hardy–Weinberg equilibrium

due to excess of homozygotes, as shown by the significantly positive FIS values

obtained. These values were consistently high among populations (mean FIS =

0.240).

Nuclear population differentiation

Most pairwise FST comparisons were significant after correction for multiple tests,

with a few exceptions involving pairwise comparison of Marchamalo (Mch) with other


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populations (Table S5). The Mantel test showed a significant pattern of isolation-by-

distance between populations with both logarithmic and original values of genetic and

geographic distances (R2 = 0.74, P < 0.0001) (Figure S1), suggesting that a model of

isolation-by-distance could explain the genetic structuring of A. iberus populations.

Samples from the Mar Menor Lagoon showed no relationship between genetic

differentiation and geographic distance (data not shown).

The Bayesian clustering procedure (Pritchard, Stephens, and Donnelly, 2000)

yielded consistent estimates of the highest likelihoods for the model with K = 5

(Figure 3). According to average proportions of membership of each pre-defined

population (Q), most specimens from the Mar Menor Lagoon were significantly

assigned to two inferred clusters (Q > 0.90). The specimens from the Alicante (Vin

and SPo) region were clustered in only one inferred group with assignment

probability higher than 0.93. The Almerian sample from Adra (Adr) constituted a

unique cluster with a high Q value. The remaining populations comprised the

remaining genetic group, although some individuals showed varying degrees of

admixture between clusters, especially the captive population of Albuixec (Alb)

(Figure 3).

Estimates of the number of migrants among populations and population decline

inference

Results obtained with BAYESASS yielded low estimates of contemporary gene flow

across sampling sites (m values ranged from 0.000 to 0.278) (Figure 4). On the other

hand, historical rate of A. iberus migrants calculated by MIGRATE revealed also low


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migration among populations (estimates of m, calculated from M, were 0.001 to

0.920) (Figure 4). Consistently, the number of migrants per generation Nm, calculated

with MIGRATE was also low (range: 0.004 to 1.113). When we statistically compared

the contemporary and historical migration rates, we observed that the m values

obtained with MIGRATE were significantly higher to the values obtained with

BAYESASS (Student’s t-test = 12.04, P < 0.0001, df = 610). Estimates of the

effective population size obtained with MIGRATE were scaled using microsatellite

mutation rates of 10-3 per locus per generation (Steinberg et al., 2002) to calculate

the mean effective population size (Ne) (Table S6). A wide range of effective

population sizes was detected among sampling sites (mean Ne ± SD; 14868 ±

18872).

The Wilcoxon test detected recent bottlenecks (P < 0.05) for all populations

when the IAM, SMM, and TPM models were assumed. The mode-shift indicator test

employed to detect genetic bottlenecks revealed distortion of allele frequency

distributions characteristic of a recent bottleneck for the majority of the populations.

Discussion

The consequences of fragmentation and restricted migration on the genetic

divergence among populations can be extreme, and may result in the increase of

population structuring and eventually in local extinctions (Frankham, 1995;

Templeton, 1990). Aphanius iberus is considered an endangered species under the

IUCN Red List (Crivelli, 2006) and the Red Book of Freshwater Fishes (Doadrio,

2001) and is protected by national and international legislation (Elvira, 1990; Elvira,

1995). Current distribution fragmented into small isolated areas, and populations


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showing a severe declining trend, primarily the result of habitat fragmentation, water

pollution, and the introduction of exotic species (Oliva-Paterna et al., 2006; Rincón et

al., 2002).

Despite its critical situation, the mtDNA genetic variability within the species

was higher than reported for its sister group, the endangered Aphanius baeticus

(Gonzalez et al., 2015; Gonzalez et al., 2014), and at the same level as reported for

non-endangered Mediterranean killifish species, e.g. Aphanius fasciatus (Ferrito et

al., 2013; Pappalardo et al., 2015; Rocco et al., 2007). Aphanius iberus shows

weaker mtDNA geographic structure compared to primary freshwater fish populations

of the Cyprinidae with similar distribution (Mesquita et al., 2005; Perdices and

Doadrio, 2001). The strong geographic structure in these populations of primary

fishes has been determined to be due to glacial refugia resulting in their restriction to

permanent riverbeds (Gante, 2011; Gomez and Lunt, 2007). In contrast, our results

suggest that Pleistocene glaciations had a lesser effect on the demographic structure

of A. iberus populations, similar to A. baeticus (Gonzalez et al., 2014) and Aphanius

farsicus (Gholami et al., 2015). Consistently, Tajima’s D, R2, and Fu’s F results

rejected the null hypothesis of constant size, as shown by the general pattern of

negative significant values obtained. Moreover, the unimodal mismatch distribution

obtained also suggested recent expansion of the A. iberian population.

The only exception to this pattern was in Villena and Sax, which showed a

high degree of mtDNA differentiation based on AMOVA tests and pairwise ΦST

comparisons when compared with other A. iberus populations. The Villena population

was also previously identified as a unique entity based on mtDNA markers


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(Fernández-Pedrosa et al., 1995; Perdices et al., 2001). A possible explanation for

this genetic pattern is the historical presence of the Villena Lagoon, of endorheic

origin related to the continentalization process that began in the late Miocene and

concluded in the early Pliocene with the disconnection of the Mediterranean and the

Atlantic zones delineating continental shelf characteristics of the Levantine region

(Estevez et al., 2004). This lagoon likely played an important role in the isolation and

consequent divergence of the populations that inhabit it. Similar levels of mtDNA

genetic differentiation detected in the closely related population of A. fasciatus have

been interpreted as historical fragmentation and restriction of gene flow (Ferrito et al.,

2013; Maltagliati and Camilli, 2000; Pappalardo et al., 2015). Several nuclear alleles

were shared with other populations within the region, including Albuixec (north

Levante), possibly indicative of secondary contact. Accordingly, no significant

differences in nuclear genetic diversity were observed between the wild and captive

populations. Given the proximity of the Villena and Sax to the Vinalopó and Santa

Pola populations, it would be expected to be more closely related to the former than

to the Albuixec population. This may indicate a complex succession of historic

contact within the north Levantine region, which sees intermittent flooding with large

discharges from rivers, outflows, canals or other watercourses allowing connectivity

and subsequent gene flow between populations. When the lagoon was drained in

1803, a canal was constructed to divert water to the Vinalopó River. This may have

promoted connection with other populations and favoured the intermixing of nuclear

alleles. Altogether, results indicate that Villena and Sax are only mitochondrically

isolated, sharing nuclear alleles with other Levantine populations, probably as a

consequence of incomplete lineage sorting (Maddison and Knowles, 2006).


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Overall FST estimates and AMOVA results based on the microsatellite data

clearly indicated significant genetic structure among A. iberus populations throughout

its distribution. Pairwise estimates of FST ranged from 0.02 to 0.39, the same level as

those reported for some endangered cyprinids (Mesquita et al., 2005). These

significant values included sites separated by 3-20 km, indicating limited current

dispersal of populations. The STRUCTURE analysis also rejected the null hypothesis

of panmixia and indicated the existence of five clusters or genetic demes of A. iberus.

The Mantel test performed on all samples detected significant positive correlation

between genetic differentiation and geographic distance. These results suggest that

a model of isolation-by-distance may explain the genetic structure pattern observed

in A. iberus. Some degree of mtDNA genetic differentiation, detected in the haplotype

network analysis, was also observed in the Catalonian samples and may support the

idea of isolation-by-distance as a source of genetic isolation. Analysis of additional

specimens from the extreme north of the species distribution is needed to further

support this hypothesis.

Habitat fragmentation is one of the factors that may prevent gene flow among A.

iberus populations. The low levels of historical and contemporary migration detected

with MIGRATE and BAYESASS+ support this hypothesis. The lower levels of

contemporary migration detected are not surprising given the anthropogenic habitat

degradation and fragmentation occurred in the last century. The results of the

equilibrium-based methods, as implemented in BOTTLENECK, also supported the

hypothesis of recent reduction in several A. iberus populations. On the other hand,

the presence of genetic admixture in individuals not in geographic proximity indicates

possible human-mediated translocation by the construction of different structures


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such as irrigation canals. This could be the case with specimens from Adra, which

show genetic similarities to those of Sax, Villena, and Albuixec, the last being

geographically most distant population from Adra. Previous studies suggest the

existence of two populations in Adra, however no separation was found in our

analysed specimens (Casas et al., 2011). The first record for the species in Adra was

from 1990 (Martinez-Vidal and Castro, 1990; Paracuellos and Nevado, 1994),

indicating possible recent origin of this population through human translocation.

Although highly speculative, the hypothesis of introduction following the construction

of irrigation canals cannot be rejected. The high number of private alleles in Adra due

to a possible recent founder event may support this inference.

In addition to its broad salinity tolerance, A. iberus has adapted to

environments highly varied in temperature and oxygen availability, such as the Mar

Menor Lagoon and the surrounding salt exploitation wetlands (Lozano, 1960; Oliva-

Paterna and Torralva, 2008). At this smaller geographic scale, the species is locally

abundant (Doadrio et al., 1996; Oliva-Paterna et al., 2006; Oliva‐Paterna et al., 2009)

despite the harshness of the environment. The populations sampled in the Mar

Menor Lagoon exhibited high levels of between-population genetic differentiation at

small spatial scales, combined with low within-population genetic diversity, lack of

isolation by distance, and reduced gene flow in recent generations. Estimated Ne

values for the Mar Menor Lagoon were lower than the census size based on field

observations (Oliva-Paterna et al., 2006). The STRUCTURE analysis defined a

group of subpopulations occupying separate patches with no geographic correlation.

For example, specimens from the Marchamalo site in the south were genetically

more closely related to the northern samples. Taken together, results suggest that


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these patterns are the result of recent drift processed due to population isolation over

the past centuries driving genetic differentiation at small spatial scales and reducing

genetic diversity within populations. This may have ultimately lead to an overall

reduction of genetic diversity for the species. Plasticity in adaptation to a changing

environment allowed species persistence by relocation to artificial habitats such as

man-made evaporation ponds and canals or to natural salt lagoons, which are

normally less suitable for such invasive species as the mosquitofish (Oliva-Paterna et

al., 2006; Verdiell-Cubedo et al., 2014). The development of more extreme

environmental conditions may have led to stronger selective pressure in these

populations, enhancing genetic drift and ultimately leading to increased genetic

structure between populations (Hrbek and Meyer, 2003; Pappalardo et al., 2015).

During the past century, anthropogenic manipulation has resulted in a large reduction

of natural habitat in the Mar Menor Lagoon (Pérez-Ruzafa et al., 2005), with

shoreline urban modification affecting fish assemblages in littoral areas (Verdiell-

Cubedo et al., 2014). The dredging and construction of channels to access the sea,

erosion due to urbanization, and expansion of salt exploitation wetlands have

resulted in changes in the morphology of the lagoon that has ultimately led to

reduction and fragmentation of A. iberus habitat. Once fragmented, the habitat

patches show reduced gene flow between the isolated and putative founded

populations.

Recent studies of the isolated Marchamalo wetland (Mar Menor Lagoon) have

reported massive mortality rates for younger A. iberus individuals corresponding with

salinity peaks (Oliva‐Paterna et al., 2009; Verdiell-Cubedo et al., 2014). Larval state

individuals and juveniles are sensitive to high salinity levels (Oliva‐Paterna et al.,


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2009) and, since inter-habitat migration relies mainly on the juvenile and adult

stages, changes in water salinity could limit species capacity for dispersal. Thus,

dispersion of individuals could be severely restricted, precluding effective

recolonization of empty patches. To reveal whether the Mar Menor populations

behave as a classic metapopulation, it will be necessary to conduct more complete

genetic surveys that include a time series of samplings to better explain the process

of dispersal and recolonization among the fragmented populations (Dobson, 2003).

Conservation of wild populations of threatened species requires genetic and

ecological management actions that facilitate recovery in natural habitat (Frankham,

Ballou and Briscoe, 2002; Mills, 2007). Elimination of a niche occupied by a species

will inevitably result in the species’ (local) extinction (Frankham, 2005). Susceptibility

to fragmentation differs with species and involves historical population size, dispersal

capacity, and historical population structure, presenting a challenge in managing

anthropogenically altered landscapes (Dixo et al., 2009). In the case of A. iberus,

habitat fragmentation has been determined as a major threat to maintaining natural

population diversity (Reichenbacher and Sienknecht, 2001). The low levels of gene

flow detected (based on Nm estimates) suggests that A. iberus has little capacity for

active dispersion, while its relatively high genetic diversity implies that its life-history

traits have contributed to genetic maintenance and conservation (Schönhuth et al.,

2003). For instance, the wide diversity of habitats in which A. iberus currently occurs

demonstrates high ability to adapt to extreme ecosystems, in comparison to the

introduced exotic species that compete for the same ecological niche, such as the

eastern mosquitofish Gambusia holbrooki (Alcaraz et al., 2008; Carmona-Catot,

Magellan and Garcia-Berthou, 2013; Oscoz, Miranda and Leunda, 2008). In


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ecogeographical areas such as the Mar Menor coastal lagoon, genetic structure

appears complex and may not adequately reflect the habitat variations in which the

species occurs. Thus, on a short-term management level, ecological features of local

areas must be taken into consideration to ensure maintenance of A. iberus

intraspecific diversity.

Funding

This work was supported by the project “Asistencia técnica para el análisis genético y

el estudio filogeográfico de las poblaciones de salinete (Aphanius iberus)” from La

Comunidad Autónoma de Murcia and by the projects of the Ministerio de Ciencia e

Innovación (CGL2010-15231_BOS and CGL2013-41375-P) to Ignacio Doadrio.

Acknowledgements

We are grateful to P. Garzón, J. L. González, and S. Perea for collection support and

to L. Alcaraz for her assistance in the laboratory. We also thank the Subject Editor

and three anonymous reviewers for their insightful comments on a previous version

of the manuscript.

Data Availability

We have deposited the primary data underlying these analyses as follows:

- Newly identified mtDNA sequences obtained in this work were deposited in

GenBank under accession numbers KU174217-KU174389 (see also Table S1).


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Table 1. Collecting localities, number of specimens of A. Iberus individuals sampled and analysed for both the nuclear (microsatellite) and mtDNA (cyt b) markers. Samples collected at the Mar Menor (MM) are also indicated.

Region Locality OCUs* Code Microsatellites Mitochondrial DNA

(Cytb)

Cataluña Aiguamolls de l'Empordà

Catalonian (I) AE 0 2

Alicante Villena Levantine (V) Vil 26 13

Alicante Sax Levantine (V) Sax 26 9

Alicante Vinalopó Levantine (V) Vin 26 12

Alicante Santa Pola Levantine (V) SPo 26 12

Valencia Albuixec Levantine (V) Alb 26 9

Murcia San Pedro (MM) Murcian (VI) SPe 24 10

Murcia Marchamalo (MM) Murcian (VI) Mch 31 11

Murcia La Encañizada (MM) Murcian (VI) Enc 25 10

Murcia La Hita (MM) Murcian (VI) Hit 20 10

Murcia Carmolí (MM) Murcian (VI) Cam 30 10

Murcia Punta Lengua de Vaca (MM)

Murcian (VI) PLV 30 6

Murcia Los Urritias (MM) Murcian (VI) Urr 24 7

Murcia La Ribera (MM) Murcian (VI) Rib 30 5

Murcia El Ciervo (MM) Murcian (VI) Cie 22 10

Murcia Playa Arsenal (MM) Murcian (VI) PA 0 2

Murcia Río Chícamo Murcian (VI) Chi 24 10

Murcia Rambla de las Moreras

Murcian (VI) RMo 22 10

Murcia Rambla de las Pinillas

Murcian (VI) RPi 6 5

Almeria Adra Murcian (VI) Adr 17 10

Total

435 173

*Based on Doadrio et al. 1996


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Table 2. MtDNA (cybb) diversity estimates and neutrality test results displayed for A. iberus. The described values are: the number of sampled individuals (N), the number of observed haplotypes (Nh), the haplotype (Hd) and nucleotide (π) diversities, with their standard deviations (SD) in brackets. All neutrality tests performed were significant (P >0.05).

Region sampled Diversity indices Neutrality test

N Nh Hd (SD) π (SD) Tajima's D Fu's Fs R2

All individuals tested 173 59 0.85 (0.03) 0.005 (0.001) -2.07* -39.81*** 0.09***

Catalonian 2 1 / / / / /

Levantine 55 25 0.91 (0.02) 0.097 (0.001) -0.46 -2.44 0.12***

Murcian 116 36 0.70 (0.05) 0.002 (0.000) -2.35** -36.39** 0.09***

Mar Menor lagoon 81 21 0.51 (0.07) 0.001 (0.000) -2.63 -19.80 1.22***


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Table 3. Summary of genetic variability estimates for the microsatellite loci tested in Aphanius iberus: number of samples (N), average number of alleles across all loci (NA), allelic richness (RS), number of private alleles (PA), observed and expected heterozygosities (HO and HE, respectively) and the mean inbreeding coefficient (Fis). Significant values are indicated in bold.

Locality Code N NA RS PA Ho He Fis

Villena Vil 26 4.1 ± 1.8 3.01 1 0.363 0.481 0.249

Sax Sax 26 3 ± 1.3 2.38 0 0.341 0.432 0.214

Vinalopó Vin 26 3.3 ± 1.3 2.71 2 0.555 0.502 -0.109

Santa Pola SPo 26 3.7 ± 0.8 2.72 0 0.330 0.437 0.249

Albuixec Alb 26 3.1 ± 0.7 2.59 0 0.330 0.469 0.301

San Pedro SPe 24 5.1 ± 2.3 3.50 1 0.387 0.544 0.293

Marchamalo Mch 31 5.7 ± 2.5 3.81 1 0.424 0.607 0.305

La Encañizada Enc 25 4.3 ± 2.7 3.06 0 0.383 0.492 0.225

La Hita Hit 20 4.7 ± 2.4 3.69 0 0.407 0.575 0.297

Carmolí Cam 30 4.9 ± 3.2 3.32 1 0.424 0.482 0.122

Punta Lengua de Vaca PLV 30 5 ± 3.2 3.47 4 0.500 0.536 0.068

Los Urritias Urr 24 5.1 ± 2.8 3.74 2 0.464 0.568 0.186

La Ribera Rib 30 4.9 ± 2.7 3.49 0 0.352 0.573 0.389

El Ciervo Cie 22 4.1 ± 2 3.20 0 0.331 0.525 0.375

Río Chícamo Chi 24 3.9 ± 2 2.89 1 0.333 0.504 0.343

Rambla de las Moreras RMo 22 3.3 ± 1 2.57 0 0.526 0.499 -0.055

Rambla de las Pinillas RPi 6 2.7 ± 1 2.71 1 0.262 0.558 0.555

Adra Adr 17 3.4 ± 1.6 2.82 3 0.336 0.479 0.305


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Figure 1.


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Figure 2.


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Figure 3.


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Figure 4.


Documents

Elena G. Gonzalez1 1,2 , Mar Torralva , and Ignacio ...digital.csic.es/bitstream/10261/197601/1/679698.pdf · Ciencias Naturales, MNCN-CSIC, José Gutiérrez Abascal, 2, Madrid, Spain,