Genetic structure of the killifish Aphanius fasciatus, Nardo 1827 (Teleostei, Cyprinodontidae), results of mitochondrial DNA analysis

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Genetic structure of the killifish Aphanius fasciatus, Nardo 1827 (Teleostei, Cyprinodontidae), results of mitochondrial DNA analysis ARTICLE in JOURNAL OF FISH BIOLOGY · MARCH 2008 Impact Factor: 1.66 · DOI: 10.1111/j.1095-8649.2007.01748.x

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Journal of Fish Biology (2008) 72, 1154–1173 doi:10.1111/j.1095-8649.2007.01748.x, available online at http://www.blackwell-synergy.com

Genetic structure of the killifish Aphanius fasciatus, Nardo 1827 (Teleostei, Cyprinodontidae), results of mitochondrial DNA analysis A. M. P APPALARDO *, V. F ERRITO *, A. M ESSINA †, F. G UARINO †, T. P ATARNELLO ‡, V. D E P INTO † AND C. T IGANO *§ *University of Catania, Department of Animal Biology ‘‘Marcello. La Greca’’, Catania, Italy, †University of Catania, Department of Chemical Science, Catania, Italy and ‡University of Padua, Department of Public Health, Comparative Pathology and Veterinary Hygiene, Padua, Italy (Received 6 March 2007, Accepted 23 October 2007) Aphanius fasciatus is a cyprinodont distributed in the salty coastal water of the central and eastern Mediterranean Sea and occasionally in internal fresh water. In this work, the authors have investigated the genetic structure of eight populations of the killifish A. fasciatus from Sardinia and Sicily. The comparison of the mtDNA control region of 237 individuals revealed a total of 49 haplotypes. Several unique haplotypes were present in each population, and no common haplotype was found among Sicilian and Sardinian populations. Almost all Sardinian populations shared a common haplotype, and indeed the four Sicilian populations examined did not share any as determined by the parsimony network analysis. The analysis of molecular variance showed that the percentage of variation among populations is much higher than within each population of A. fasciatus. The overall FST value is very high (0
78) and supports an extensive genetic structure of the populations. The observed genetic differentiations of A. fasciatus populations were discussed taking into account the palaeogeographic and palaeoclimatic events that interested the Mediterranean area from Miocenic to Pleistocenic age. The results provide new insight into the knowledge of the pattern of genetic structure and of # 2008 The Authors evolutionary processes occurring in this species. Journal compilation # 2008 The Fisheries Society of the British Isles

Key words: Aphanius fasciatus; control region; gene flow; intraspecific variation; mtDNA.

INTRODUCTION The population structure of brackish-water species has attracted many investigations. These species live in environments characterized by rapid and wide changes of both physical–chemical parameters (Cognetti, 1994; Cognetti & Maltagliati, 2000), thus peculiar evolutionary mechanisms have been detected in brackish-water fishes (Bernardi, 2000; Bernardi & Talley, 2000; Bernardi et al., 2003; Martins et al., 2003; Patarnello et al., 2003; Abila et al., 2004; Rivera et al., 2004; Domingues et al., 2005; Bowen et al., 2006). However, the genetic §Author to whom correspondence should be addressed. Tel.: þþ30 095 7306030; fax: þþ39 095 327990; email: [email protected]

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drift, inbreeding and founder effect may produce loss of genetic diversity at an intraspecific level and may drive population differentiation at both morphological and genetic levels (Hartl & Clark, 1997). To evaluate the opposite effects of the intraspecific homogenization in quickly evolving closed environments, the authors decided to study populations of the killifish Aphanius fasciatus, Nardo 1827. The killifish A. fasciatus lives in brackish-water habitat, such as ponds and coastal lagoons, it is relatively sedentary and produces large benthic eggs; the adults have limited migrating capabilities (Maltagliati, 1998). Recent investigations on intraspecific differentiation of this species pointed out some discrepancy between the genetic uniformity v. the genetic divergence of the populations of this species (Maltagliati,1999, 2002; Hrbek & Meyer, 2003; Tigano et al., 2004). At a morphological level, Villwock (2004) described a remarkable uniformity in morphometric as well as in certain reproductive patterns in populations of A. fasciatus. On the contrary, statistically significant osteological differentiation were discovered by Tigano et al. (2001, 2004) and Ferrito et al. (2007) among 14 populations of this species. These last results might be explained by an occasional gene flow, by genetic drift or natural selection or by some of these forces acting together to shape the gene pool of the species (Cimmaruta et al., 2003). The aim of this work was to present, for the first time, an analysis of the genetic structure of killifish A. fasciatus populations at molecular level. The authors have selected killifish populations in Sardinia and Sicily, the major islands of the Mediterranean Sea because of their very interesting palaeogeographic history. The molecular marker studied in this work is the left domain of the mtDNA control region. The 59-end of the control region shows the highest observed rates of base substitutions and insertion/ deletion events in vertebrates (Saccone et al., 1987). This rapid mutation rate is at least five times higher than the remaining mitochondrial genome (Brown et al., 1979). The predominantly maternal inheritance of vertebrate mtDNA provides a useful tool to evaluate evolutionary genetic divergence (Wilson et al., 1985). The mtDNA control region comprises a central conserved sequence flanked by highly variable right and left domains. These domains are among the most popular markers for evolutionary relationships among populations (Bernardi, 2000). Aphanius fasciatus is a species included in the ‘World Conservation Union (IUCN) Red list of threatened species’: former studies by our group (Ferrito & Tigano, 1996) showed that several populations were already extirpated in Sicily; therefore, an understanding of the population genetic variation of this species is important for a successful conservation management.

MATERIALS AND METHODS SAMPLE COLLECTION AND DNA EXTRACTION A total of 237 adult individuals were sampled from six geographical locations of Sardinia and four of Sicily. Data from Sicilian populations of Longarini (LO), Foce Marcellino (FM), Salina Curto Marsala (SMA) and Salina Chiusa Trapani (TP) previously studied by Tigano et al. (2004, 2006) were also used in the statistical analyses (Table I). Total genomic DNA was extracted from muscle tissue (25–30 mg) using DNeasy tissue kit (Qiagen, Milan, Italy) following the manufacturer’s instructions. # 2008 The Authors Journal compilation # 2008 The Fisheries Society of the British Isles, Journal of Fish Biology 2008, 72, 1154–1173

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TABLE I. List of the populations of Aphanius fasciatus examined in this work; sampling areas, population identification code and number of specimens examined Groups Sicily

Sardinia

Sampling areas Longarini* Longarini Foce Marcellino* Foce Marcellino Salina Curto Marsala† Salina Curto Marsala Salina Chiusa Trapani† Salina Chiusa Trapani Santa Giusta Pauli Figu Pauli Majori Marceddı` S’Ena Arrubia Santa Gilla

Collection period May 2005 May 2005 June 2006 June 2006 June 2003 June 2003 June 2003 June 2003 June 2003 July 2004

Code

Specimens (n)

LO LO FM FM SMA SMA TP TP SG PF PM MR SR SL

10 13 10 13 9 15 10 15 23 25 24 23 24 23

*These sequences were reported in Tigano et al. (2004). †These sequences were reported in Tigano et al. (2006).

DNA AMPLIFICATION AND AUTOMATED SEQUENCING The 59-end of the mitochondrial control region of the killifish specimens was amplified using the following primers: reverse primer, 59-CCTGAAGTAGGAACCAGATG-39 (Meyer et al., 1990); forward primer, 59-ACTATTCTTTGCCGGATTCTG-39 (Tigano et al., 2004). Polymerase chain reactions (PCRs) were carried out in 50 ml. Each reaction contained 0
5 mM of each primer, 0
2 mM deoxynucleoside triphosphate, 1
5 mM MgCl2, 1 PCR buffer, 1 U of Taq polymerase (Invitrogen, Milan, Italy) and 50– 100 ng of genomic DNA. The PCR conditions were as follows: initial denaturation at 94° C for 2 min, followed by denaturation at 94° C (30 s), annealing at 48° C (30 s) and the extension at 72° C (40 s) repeated for 35 cycles and by a final extension at 72° C for 7 min. Amplified DNA fragments were purified using a QIAquick PCR Purification kit (Qiagen). Sequencing was performed upon both strands using an ABI Prism 3100 automated sequence (Applied Biosystems, Foster City, CA, U.S.A.). All sequences are available under the GenBank (haplotypes accession numbers are reported in Appendix II).

SEQUENCE DATA ANALYSIS Sequences were aligned using ClustalX (Thompson et al., 1997). Ambiguous regions of the alignment were systematically identified and removed using the programme GBlocks v.091b (Castresana, 2000). The default programme parameters were used, exclusive of allowing a minimum block length of 5 and gaps in 50% of positions. Genetic variability was estimated using two parameters: nucleotide diversity (p) described as the average number of nucleotide differences per site between two sequences, and haplotype diversity (h). An analysis of molecular variance (AMOVA), implemented in the programme Arlequin ver. 3.0 (Excoffier et al., 2005), was applied to the distance matrix to estimate variance components and population pair-wise distance

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measures (FST). The analyses of genetic variance were performed with the populations separated into two distinct groups: those from Sicily, on one hand (LO, FM, SMA and TP) and those from Sardinia [Santa Giusta (SG), Pauli Figu (PF), Pauli Majori (PM), Marceddı` (MR), S'Ena Arrubia (SR) and Santa Gilla (SL)] on the other. FST values were estimated both for population pairs and for all populations, using a ‘weighted’ analysis of variance (Weir & Cockerham, 1984), and taking into account genetic distances between population pairs (Excoffier et al., 1992). Measures of substitution saturation were performed using DAMBE 4.2.13 (Xia, 2000; Xia & Xie, 2001) for mitochondrial control region sequences. Phylogenetic relationships among sequence haplotypes were examined using neighbour-joining (NJ) and maximum parsimony (MP) analyses with Aphanius danfordii (Boulenger, 1890) (GenBank accession number U06062) designated as the outgroup. Distance method (NJ) was used throughout for representing haplotype relationships. Phylogenetic analysis was performed using MEGA 2.1 (Kumar et al., 2001). Pairwise divergence was calculated using a Kimura two-parameter (K2P) algorithm in MEGA. The robustness of internal branches of distance was estimated by bootstrapping (Felsenstein, 1985) with 1000 replicates. A heuristic search (with 10 replicates of random addition of sequences) was performed in PAUP ver 4.0b10 (Swofford, 2002) assigning an equal mass to all characters. The relationships between unique control region haplotypes were described with a parsimony network generated with the programme TCS ver. 1.13 (Clement et al., 2000).

RESULTS SEQUENCE ANALYSIS

A 378 bp portion of the mitochondrial control region was sequenced in 237 individuals of A. fasciatus from 10 Mediterranean Sea populations (Table I). The base composition showed an A–T bias (A þ T content ¼ 0
67) as usual for the D-loop or control region. Out of these 378 bases, 55 were variable and 38 were parsimony informative (Appendix I). Saturation analysis demonstrated that the mitochondrial control region data set contains little substitution saturation [the observed index of substitution saturation (Iss) values are significantly lower than the critical index of substitution saturation (Iss.c)]; thus, our data could be well used for phylogenetic analyses. The slope of the curves [the plot shows transversions/transitions v. divergence (K80 genetic distances calculated by DAMBE)] (figure not shown) also points out the strength of the sequence data. A comparison of all investigated sequences led to the identification of 49 distinct mtDNA haplotypes. Almost all haplotypes are population-specific, among them, 10 distinct mtDNA haplotypes were found exclusively in the two eastern Sicilian populations; two haplotypes were found that are shared by the Sardinian and western Sicilian populations (Appendix II).

P H Y L O G E N E T I C R EL A T I O N S H I PS

The phylogenetic trees constructed with the NJ method (K2P model) and the MP method were superimposable and revealed that the Sicilian population LO is separated from all the other populations positioned at the base of the tree (Fig. 1). The second node isolates the second eastern Sicilian population # 2008 The Authors Journal compilation # 2008 The Fisheries Society of the British Isles, Journal of Fish Biology 2008, 72, 1154–1173

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analysed (FM) from a cluster containing all of the Sardinian populations and the western Sicilian populations. Among Sardinian populations, MR and SR form two clusters that are separated from the others; the relationships among the remaining Sardinian populations and the western Sicilian populations are not well resolved. The NJ tree was supported by the parsimony network analysis that produced two haplotype groups (Fig. 2). The first group includes all haplotypes of LO, completely separated by all the other populations. The second group includes all Sardinian haplotypes and the remaining Sicilian haplotypes. Within this group, h12 is the most common haplotype, shared between all populations, but SR; the Sicilian FM haplotypes were separated by at least six mutational steps from the haplotype h29 shared by some Sardinian populations and from h19 shared by the western Sicilian populations. Moreover, the relationships within this second group revealed several reticulated connections, indicating a substantial amount of parallel substitutions. These two groups could not be connected together under the default confidence limit of 95% (Templeton et al., 1992).

P O P U L A T I O N S T R U C TU R E A N D G E N E T I C DI V E R G E N C E

The genetic variation within each population was described as haplotype diversity (h) and nucleotide diversity (p) indices (Table II). The large range of haplotype numbers led to a high haplotype diversity (from 0
62 to 0
82) for almost all populations. The Sicilian population FM showed the lower values of haplotype and nucleotide diversity (h ¼ 0
24 and p ¼ 0
07  102) (Table II). In the AMOVA, only the 13
78% of the molecular variance was attributable to variations among groups and the 64
44% to variations among populations within groups; the 21
78% of the molecular variance was related to variations within the same population. The FST value (0
78) (Table III) is statistically significant with respect to the null hypothesis of panmixia. As expected from the phylogenetic relationships described above, the pair-wise comparison of FST values showed that high values were found between Sardinian and Sicilian populations (average FST ¼ 0
60). Among Sardinian populations, the lowest FST values were found between PF/PM (FST ¼ 0
01), PF/SG (FST ¼ 0
03) and PM/SG (FST ¼ 0
06) and the highest values between SR/SL (FST ¼ 0
39). Very high FST values were found between the Sicilian populations FM/LO (FST ¼ 0
91), indeed the lowest FST values were found between SMA/TP (Table IV). The average sequence divergence matrix calculated according to the K2P model among Sardinian and Sicilian populations shows different values for

FIG. 1. Neighbour-joining phylogeny and spatial distribution of the 49 different mtDNA haplotypes identified within the sequenced part of the control region (378 bp). Estimations of genetic divergences were calculated on the basis of the two parameters model of Kimura (1980). The numbers at each node indicate percentage recovery (>50%) of the particular nodes (1000 bootstraps replicates). The arrows indicate the shared haplotypes. The asterisk indicates the Sicilian populations studied by Tigano et al. (2004, 2006). Sicily south-eastern: FM, Foce Marcellino; LO, Pantano Longarini; SMA, Salina Curto Marsala; TP, Salina Chiusa Trapani. Sardinia: MR, Marceddı` ; PF, Pauli Figu; PM, Pauli Majori; SG, Santa Giusta; SL, Santa Gilla, SR, S'Ena Arrubia.

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LO and FM (eastern Sicily) and SMA and TP (western Sicily). In particular, the percentage of sequence divergence between Sardinian populations and LO on one hand and FM on the other is 4
29 and 2
13%, respectively. A very low average sequence divergence (0
33%) is found between Sardinian and western Sicilian populations. Moreover, within Sardinian populations, the average sequence divergence is 0
40%, while the Sicilian populations show an average sequence divergence of 2
56%. This value increased to 4
00% when the western Sicilian populations were compared with the eastern population LO.

DISCUSSION In this study, the authors provide clear evidence for significant genetic differentiation among the populations of the killifish A. fasciatus both at regional (between Sardinian and Sicilian populations) and at local scale (between different Sicilian populations). The FST-based population differentiation was statistically significant (FST ¼ 0
78). The FST values in pair-wise comparisons among adjacent Sardinian populations (PF, PM, SG), western Sicilian populations (SMA, TP) and between these two groups of populations were not significant. Studies by Brown & Chapman (1991) found low values of FST ¼ 0
04 in populations of Fundulus heteroclitus (L.) distributed along the Atlantic coast of the U.S.A. According to Wright (1978), the values of FST >0
25 indicate a very great genetic differentiation. High FST values are usually typical in freshwater species occupying unconnected lake habitats where the authors have very limited scope for gene flow (Ward et al., 1994). Studies by Bernardi & Talley (2000) indicated high FST values in populations of the Cyprinodontiformes Fundulus parvipinnis Girard, 1854 (FST ¼ 0
70), a species living in environments similar to those preferred by A. fasciatus. It is well known that mitochondrial FSTs are usually larger than nuclear FSTs. This is perhaps due to the different evolutionary dynamics of the two genomes (Slade et al., 1998). The authors suppose that the high FST values observed in this work could result from long independent histories among populations that are currently proximate to one another but have been isolated historically. The hypothesis is supported by studies by Villwock (1982) which proved, with crossbreeding experiments, that populations of others species of the genera Aphanius and Cyprinodon represent different populations belonging to different interfertility groups within the same species, or in other words, reflecting different stages of species ‘in statu nascendi’. To solve these taxonomic problems, it will be necessary to test our hypothesis with the nuclear markers. The high number of unique haplotypes per population (only six haplotypes out of 49 were shared: two between Sardinian populations, two between Sicilian populations and two among Sicilian and Sardinian populations) indicates differentiation among sites. The genetic differentiation shown in this work could be interpreted by taking into account the palaeogeographic and palaeoclimatic history of the Mediterranean area. During the Quaternary, A. fasciatus populations were subjected to the geological and climatic changes borne by the coastal areas where the killifish lived. According to Villwock (2004), the speciesgroup A. fasciatus (that derived from a hypothetical common A. fasciatus/Aphanius

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FIG. 2. Parsimony network analysis of the 49 mtDNA haplotypes using TCS (Clement et al., 2000). Sicily: FM, Foce Marcellino; LO, Pantano Longarini; SMA, Salina Curto Marsala; TP, Salina Chiusa Trapani. Sardinia: MR, Marceddı` ; PF, Pauli Figu; PM, Pauli Majori; SG, Santa Giusta; SL, Santa Gilla, SR, S'Ena Arrubia.

dispar (Ruppel, 1829) ancestor) should have formed in the Lower Miocene as ¨ a consequence of the ongoing regression of the Tethys Sea. The Messinian Salinity Crisis might have allowed temporary contacts along the shoreline of the Mediterranean Sea as a result of the water level lowering (Maldonado, 1985; Margalef, 1985). However, the exact period when A. fasciatus first appeared is unknown or it is known if an ancestral Aphanius pliocenii existed (J. Gaudant, pers. comm.). Furthermore, studies by Hrbek & Meyer (2003) show that A. fasciatus is the only species of the genus that does not fit the hypothesis of Messinian vicariant differentiation and that the diversification of its populations started c. 5 million years ago (the oldest population was that from Lake Bafa in Turkey showing a time of separation corresponding to 3
99 million years). At a regional scale, the presence of shared haplotypes among the Sardinian and western Sicilian # 2008 The Authors Journal compilation # 2008 The Fisheries Society of the British Isles, Journal of Fish Biology 2008, 72, 1154–1173

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TABLE II. Haplotype numbers (n), haplotype diversity (h) and nucleotide diversity (p) of the populations examined in this work. The parameters reported in this table were calculated with Arlequin ver. 3.0 (Excoffier et al., 2005). The S.D. are reported in parentheses Population

Code

n

Foce Marcellino Longarini Salina Curto Marsala Salina Chiusa Trapani Marceddı` Pauli Figu Pauli Majori Santa Gilla Santa Giusta S'Ena Arrubia

FM LO SMA TR MR PF PM SL SG SR

3 7 10 6 9 9 4 9 7 8

p (102)

h 0
24 0
77 0
75 0
62 0
76 0
68 0
47 0
82 0
71 0
82

(0
11) (0
07) (0
09) (0
10) (0
09) (0
10) (0
10) (0
06) (0
09) (0
05)

0
07 0
81 0
35 0
30 0
74 0
34 0
13 0
58 0
43 0
70

(0
09) (0
49) (0
25) (0
22) (0
45) (0
25) (0
13) (0
37) (0
30) (0
43)

killifish populations denote that the Pleistocenic events would have allowed extensive gene flow within populations. Sardinia and Sicily started to separate during the Miocenic age after the Oligocenic split of the Tirrenide subcontinent (La Greca, 1989). During the Pliocenic age (from 5 to 1
7 million years ago), Sicily consisted of two islands separated from the nearby land masses: one, to the north, that included northern Sicily (equivalent to today’s Sicilian provinces of Trapani, Palermo and Messina) deriving from Tirrenide split and a smaller one that comprised the present Iblean region (equivalent to today’s Sicilian provinces of Ragusa and Siracusa) deriving from the African plate (Fig. 3). This isolation was interrupted in the Mindel–Riss interglacial (120 000 years ago) when the rejoining with the northern part of Sicily occurred (Blanc, 1942; Pasa, 1953; La Greca, 1957). Both NJ and MP trees and parsimony network analysis supported the high differentiation of the Sicilian population LO, belonging to the Southern Iblean region, that forms an obvious geographical cluster in the NJ tree. LO population’s haplotype shows no connection to the others in the network. Moreover, based on the divergence indices obtained by the control region sequences, the Sicilian LO represented the most divergent population since it presented higher genetic distance compared with the other populations analysed (average pair-wise divergence 4
0%). According to the molecular clock calibration estimates for mtDNA control TABLE III. Analysis of molecular variance calculated for the populations examined in this work with Arlequin ver. 3.0 Source of variation

d.f.

Sum of squares

Variance component

Percentage of variation

F-statistic

Among groups Among populations within groups Within populations

1 8

97
383 386
610

0
43001 Va 2
01113 Vb

13
78 64
44

FCT ¼ 0
14 FST ¼ 0
78

227

154
286

0
67968 Vc

21
78

FSC ¼ 0
75

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TABLE IV. Population pair-wise FST-values calculated with Arlequin ver. 3.0. Boldface P < 0
05 after sequential Bonferroni correction

FM LO SMA TR MR PF PM SL SG SR

FM

LO

SMA

TR

MR

PF

PM

SL

SG

0
91 0
86 0
91 0
88 0
92 0
95 0
92 0
88 0
86

0
87 0
88 0
87 0
89 0
90 0
89 0
87 0
86

0
01 0
16 0
02 0
01 0
10 0
05 0
30

0
17 0
03 0
04 0
10 0
05 0
34

0
17 0
21 0
20 0
16 0
26

0
01 0
10 0
03 0
33

0
15 0
06 0
34

0
10 0
39

0
31

SR

region (Stepien et al., 2001), sequence divergences calculated by pair-wise genetic distance (K2P) in fishes commonly ranged from 2% per MY (a slow clock) to 10% per MY (a fast clock). Assuming this range, the Sicilian population LO diverged from Sardinian populations for 429 000–2 145 000 years. Both these divergence times are compatible with the palaeogeographic events occurring in the Mediterranean basin at Pliocenic–Pleistocenic age. Therefore, the pronounced differentiation of the killifish population LO (from the other populations studied here) can be explained in terms of the relatively long period of isolation of the Iblean region. The other Sicilian population FM (located in the northern Iblean region) showed a very low index of haplotype and nucleotide diversity (h ¼ 0
24  0
11; p ¼ 0
0007  0
0009). This could suggest a relatively small effective population size. At a local scale, the two Sicilian populations showed an average pair-wise sequence divergence value of 2
86% suggesting a time of divergence at a local scale comparable with that at a regional scale (the two populations were differentiated for 286 000–1 430 000 years). This differentiation can be interpreted in the light of the palaeogeographic events that took place in the Iblean region in the lower Pleistocene (c. 1
7 million years

FIG. 3. Sicily in the lower Pleistocene (From La Greca, 1957 modified).

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ago) when a marine catastrophe took place in the north of Syracuse and in the south-eastern Iblean area (Tigano et al., 2004). Interestingly, the FM population is connected with the cluster containing all Sardinian populations and two western Sicilian populations, as showed in the NJ tree. The parsimony network analysis indicated that the FM haplotypes were separated by at least six mutational steps from haplotype h29 shared by Sardinian populations of Marceddı` and S’Ena Arrubia and the haplotype h19 shared by Sicilian populations of SMA and TP. Also, the average pair-wise divergence between FM and the Sardinian populations was 2
13% corresponding to a divergence time ranging between 213 000 and 1 065 000 years. The connection between h29 haplotype shared among Sardinian populations and the haplotypes of Sicilian populations living in FM, SMA and TP could also be explained as the result of the fragmentation of a macropopulation that was related to the oscillating movements of the sea level during the Pleistocene. Conversely, within Sardinian populations of A. fasciatus, there was a high genetic similarity (average pair-wise divergence was 0
4% and the time of divergence ranges between 20 000 and 40 000 years) based on the indices of nucleotide divergence in the mtDNA control region showed in this work. This was not unexpected; these three water bodies indeed derived by the progressive reduction of the ancient Santa Giusta lake; and both the water bodies of PF and PM are connected with SG by a regulated water channel. All Sardinian haplotypes are connected through the haplotype h12. In conclusion, Mediterranean killifishes could be an interesting model of rapid allopatric divergence, as several studies have shown at a morphological and genetic level (Maltagliati, 1999; Tigano et al., 1999, 2001, 2004; Ferrito et al., 2003). In a recent work, Ferrito et al. (2007) showed that Sardinian and Sicilian populations studied here differed in several osteological characteristics, although the underlying genetic bases for these phenotypic traits are largely unknown. Moreover, the authors cannot exclude the possibility that the influence of environmental factors could play an important role in the morphological differentiation of populations. In this study, a wide range of sequence divergence (0
12–4
73%) was found among the populations of A. fasciatus. In particular, a high sequence divergence was detected both at regional (between Sardinian and Sicilian populations) and at local scale (between the four Sicilian populations). It is noteworthy that a very small-scale genetic differentiation in this species was already revealed by Maltagliati et al. (2003) and also Cimmaruta et al. (2003) showed that in some cases genetic relationships among populations of A. fasciatus were independent of the geographic distances between samples. The results here reported provide insight into the microevolutionary processes in killifishes. On the basis of the mtDNA sequences analysed they are conclusive, but a comparison with nuclear markers is desirable. Furthermore, the authors think that the results could be relevant to conservation purposes: in order to keep the total evolutionary potential of this species, many individual killifish populations, demographically independent, as described here, should be preserved. We wish to thank A. Lussen from the Zoologisches Institut und Zoologishes Museum, University of Hamburg, for his comments that significantly improved the quality of the paper and to J. Gaudant from the Museum National d’Histoire Naturelle,

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Laboratoire de Paleontologie, CNRS, Paris. C.T. acknowledges the University of Catania (PRA 2004) and V.D.P. acknowledges the University of Catania, MIUR COFIN and FIRB2003_RBNE03PX83 for financial support. This research is part of A.M.P.’s doctoral dissertation in Evolutionary Biology on intraspecific variation of killifish Aphanius fasciatus. It is also part of C. Tigano’s research programme on the microevolutionary processes in Cyprinodontids species.

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# 2008 The Authors Journal compilation # 2008 The Fisheries Society of the British Isles, Journal of Fish Biology 2008, 72, 1154–1173

h1 h2 h3 h4 h5 h6 h7 h8 h9 h10 h11 h12 h13 h14 h15 h16 h17 h18 h19 h20 h21 h22 h23 h24 h25 h26

Haplotype

Journal compilation

T T T T T T T

A

T T T T T T T T T T T T T T T T T T T T T T T

C

T T T T T T T T T T T T T T T T T T T T T T T

C

A

G A

T T T T T T T

A

A

G

T

T

T

#

G

G G G G G G G G

A

A

A

C C C C C C C

T

A

G A

A

T

T T T T T T T T T T T T T T T T T T T T T T T

C

C

C

T

A

A A A A A A A

C

T T T T T T T

C

T

T

A

C

C C

T

T

T

A

12 13 17 39 41 52 60 75 80 88 95 96 104 106 107 109 113 118 122 127 131 136 137 138 142 143 144

Nucleotide position in the mitochondrial control region*

APPENDIX I. Variable sites of 49 mitochondrial haplotypes of Aphanius fasciatus populations

1168 A. M. PAPPALARDO ET AL.

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h27 h28 h29 h30 h31 h32 h33 h34 h35 h36 h37 h38 h39 h40 h41 h42 h43 h44 h45 h46 h47 h48 h49

Haplotype

T T T T T T T T T T T T T T T T T T T T T

T

T T T T T T T T T T T T T T T T T T T T T T T

T T T

C C C

T

G

A

C

G G

G

G

G G

G

C

T T T T T T T T T T T T T G T T T T T T T T T C

12 13 17 39 41 52 60 75 80 88 95 96 104 106 107 109 113 118 122 127 131 136 137 138 142 143 144

Nucleotide position in the mitochondrial control region*

APPENDIX I. Continued

P O P U L A T I O N S T R U C T U R E I N A P H A N I U S FA S C I AT U S

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h1 h2 h3 h4 h5 h6 h7 h8 h9 h10 h11 h12 h13 h14 h15 h16 h17 h18 h19 h20 h21 h22 h23 h24 h25 h26

Haplotype

A

Journal compilation

C C C C C C C

A

A

#

A

A

G

G G G G G

A

G

G G

A

C C

T

C C A C A A A

T

T T T T T T T

A

T

C

C

A A A A A A A A

G

G G G G G G G A A A A A A A G A A A A A A A A

C

C C C C

C C C C C C C C C C C

T

A

C C C C C C C

C C C C C C C C

T

T

C

C

A

G

C

T

G G G G G G G

A

A

C

C

G

T T

C

G

G G G

T

A

T T T T T T T T T T T T T T T T T T T T T T T

C

156 166 172 173 177 184 188 190 191 218 223 224 256 258 259 260 286 301 309 326 342 343 347 352 358 364 365 368 369

Nucleotide position in the mitochondrial control region*

APPENDIX I. Continued

1170 A. M. PAPPALARDO ET AL.

# 2008 The Authors 2008 The Fisheries Society of the British Isles, Journal of Fish Biology 2008, 72, 1154–1173

T

A A A A A A A A A A A A A A A A A A A A A A A C C C C C C C C C C C A C C C C C C C C

C C

T

C C C C C C C C C C C C C C C C C C C C C C C T

T

A

T

T

C C

C C C C

T T

T T T T T

A

G G

T T T T T T T T T T T T T T T T T T T T T T T

156 166 172 173 177 184 188 190 191 218 223 224 256 258 259 260 286 301 309 326 342 343 347 352 358 364 365 368 369

Nucleotide position in the mitochondrial control region*

*The position refers to the sequence of the haplotype h1.

h27 h28 h29 h30 h31 h32 h33 h34 h35 h36 h37 h38 h39 h40 h41 h42 h43 h44 h45 h46 h47 h48 h49

Haplotype

APPENDIX I. Continued

P O P U L A T I O N S T R U C T U R E I N A P H A N I U S FA S C I AT U S

1171

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h1 h2 h3 h4 h5 h6 h7 h8 h9 h10 h11 h12 h13 h14 h15 h16 h17 h18 h19 h20 h21 h22 h23 h24 h25 h26

Haplotype

AJ605322 AJ605323 AJ605324 AJ605329 AJ605327 AJ605328 AM183190 AM183192 AM183193 AM183194 AM884565 AM184188 AM884566 AM183202 AM884567 AM884568 AM184198 AM184199 AM184200 AM184201 AM884569 AM884570 AM184197 AM183168 AM183191 AM183195

GenBank accession numbers 20 1 2

MA

5 1 10 2 2 1 2

LO

2 12 1 3 1 1 1 1 1 1

SMA

Journal compilation

#

2 1 4

1

15 2

TP

5

3

3 2 1

16

PF

12

MR

Location

APPENDIX II. Haplotypes distribution in population of Aphanius fasciatus

18

PM

18

SL

2

12

SG

8

SR

1172 A. M. PAPPALARDO ET AL.

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h27 h28 h29 h30 h31 h32 h33 h34 h35 h36 h37 h38 h39 h40 h41 h42 h43 h44 h45 h46 h47 h48 h49

Haplotype

AM183196 AM183197 AM183199 AM183201 AM183204 AM183205 AM183206 AM183207 AM183208 AM183209 AM183210 AM183214 AM183169 AM183170 AM183174 AM183175 AM183176 AM183177 AM183178 AM183179 AM183180 AM183181 AM183182

GenBank accession numbers MA

LO

SMA

TP

APPENDIX II. Continued

2 1 1 1

MR

Location

1 1 1 1 1 1

PF

1

PM

3 1 1

SL

3 1 1 1

3

SG

1 2 1 4 7

1

SR

P O P U L A T I O N S T R U C T U R E I N A P H A N I U S FA S C I AT U S

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