Molecular Phylogenetics and Evolution 61 (2011) 1–11
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Fine-scale genetic structure, phylogeny and systematics of threatened crayfish species complex S. Chiesa a,b, M. Scalici a, R. Negrini c, G. Gibertini a, F. Nonnis Marzano b,⇑ a
Department of Biology, University of ‘‘Roma Tre’’, V. le G. Marconi 446, 00146 Rome, Italy Department of Evolutionary and Functional Biology, University of Parma, V. le Usberti 11, 43100 Parma, Italy c Institute of Zootechnical Sciences, Catholic University of Sacred Heart, Via Emilia Parmense 84, 29100 Piacenza, Italy b
a r t i c l e
i n f o
Article history: Received 25 June 2010 Revised 15 March 2011 Accepted 30 March 2011 Available online 19 April 2011 Keywords: Molecular evolution Phylogenetics Austropotamobius pallipes AFLP COI
a b s t r a c t Systematic uncertainties in the crayfish Austropotamobius pallipes are well grounded by the number of species and subspecies described using different approaches, causing scientists to define this taxon as ‘‘complex’’. However, a key task that conservation programmes are facing regarding the recent and drastic decline of European populations, is the coherent systematic classification of this threatened species. Here we present results obtained by coupling mtDNA and genome analysis suggestive of a novel evolutionary framework to explain the relationships among phylogenetic lineages of A. pallipes. The direct sequencing of mtDNA COI gene fragment revealed a strong geographic structure with four distinct haplogroups separated by a range of 5–25 mutations. However, mitochondrial data were not supported by genomic fingerprinting based on 535 AFLP polymorphisms. Nuclear markers showed an unexpected moderate level of genetic differentiation and the absence of any geographic structure. Consequently, this study proposes that the taxonomic hypothesis of a single species of A. pallipes settling the Italian continental waters, is affected by complex evolutionary events. To solve the paradox, we hypothesized an evolutive scenario in which the separation of ancient mtDNA lineages likely occurred before the latest glacial periods. However, the speciation process remained incomplete due to secondary intensive postglacial contacts that forced the mingling of the genomes, and confounds the phylogeographic signature still detectable within mtDNA. Postglacial dispersion and the following demographic events, such as founder effects, drift and bottlenecks, abruptly depleted the local mtDNA variation, and shaped the current genetic population structure of white-clawed crayfish. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction In the last decade a number of studies investigated the taxonomy and evolutionary relationships among phylogenetic lineages of crayfish species (Fezner and Crandall, 2003; Buhay and Crandall, 2005; Song et al., 2008). Although based on several different molecular approaches, the proposed taxonomic and systematic models do not appropriately fit with the observed genetic variability. Controversial data induced the scientists to describe a considerably high number of different ‘groups’ or lineages and to temporarily define crayfish species as a ‘‘complex’’ (Austin, 1996; Santucci et al., 1997; Mathews et al., 2008) or cryptic species. This means that the evolutionary and biogeographic histories of these decapods are far from being completely understood (Crandall
⇑ Corresponding author. Fax: +39 0521 905657. E-mail addresses:
[email protected] (S. Chiesa),
[email protected] (M. Scalici),
[email protected] (R. Negrini),
[email protected] (G. Gibertini),
[email protected] (F. Nonnis Marzano). 1055-7903/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2011.03.031
et al., 2000; Scholtz, 2002; Fezner and Crandall, 2003; Buhay and Crandall, 2005). Phylogeny and adaptive radiation of the family Astacidae in European regions have been interpreted as the result of past limited speciation events. Only five native European species were described up to now (Holdich, 2002; Machino and Holdich, 2006), as a result of relict taxa. The systematic uncertainty also suggests that elucidating phylogeographic relationships in this group is a pressing issue, especially regarding the implications for its conservation (Mathews et al., 2008). In this context, the white-clawed crayfish Austropotamobius pallipes (Lereboullet, 1858) represents a great example of such problems. It is the most common native river dwelling crayfish in Italy, once widely distributed in Western Europe (Souty Grosset et al., 2006), however nowadays it is suffering a progressive and drastic decline of both biomass and number of populations due to ecological and environmental factors (Gherardi and Holdich, 1999; Holdich et al., 1999; Souty Grosset et al., 2006). It is considered ‘‘vulnerable’’ in the IUCN Red List, ‘‘critically endangered’’ in the IUCN Italy, included in Annex II and V of EU Habitats Directive
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(92/43/EEC) and in Annex III and IV of Bern’s Convention. With more than 50 years of research, from Bott (1950) to Fratini et al. (2005), description of A. pallipes comprised of a maximum of four species and six subspecies (Starobogatov, 1995) to a minimum of one species (Trontelj et al., 2005) (Table 1). Accordingly, the nomenclature has been revised several times increasing the uncertainties (see Manganelli et al., 2006) and negatively impacting its conservation. Until now, the sequencing of mitochondrial genes has been the most common approach to investigate A. pallipes phylogeny, phylogeography and systematics (Grandjean et al., 2002; Fratini et al., 2005; Trontelj et al., 2005; Zaccara et al., 2005; Diéguez-Uribeondo et al., 2008; Stefani et al., 2011). However, studies based exclusively on mitochondrial data reflect the maternal pattern of inheritance, only providing a partial view of the complex phylogeographic patterns of white-clawed crayfish. Many recent studies show the contrast between mitochondrial and nuclear signal, in different zoological groups (Shaw, 2002; Koblmüller et al., 2007; Babbucci et al., 2010; Yang and Kenagy, 2009; Leaché, 2010; Rato et al., 2010): discrepancy between mitochondrial and
nuclear markers emerged when reconstructing the history of a species, extending the conflicts also in strongly support mtDNA clades. Concerns include polymorphisms in common ancestral populations, male-biased gene flow, selection on mtDNA, gene flow following hybridization and transfer of mtDNA to the nucleus (Moritz and Cicero, 2004). This make usefulness of single mitochondrial analysis controversial (Dasmahapatra and Mallet, 2006). Here we utilized for the first time an integrated approach, combining the effectiveness of COI sequences in phylogeographic analysis of genus Austropotamobius (Trontelj et al., 2005; Zaccara et al., 2005; Diéguez-Uribeondo et al., 2008; Stefani et al., 2011), with the well documented potential of the high throughput AFLP technique to assess genetic diversity within and among closely related taxa (Mendelson and Shaw, 2005; Maldini et al., 2006; Koblmüller et al., 2007). Present results based on the integration of mitochondrial data with nuclear markers challenge previously proposed hypothesis on the taxonomy of white-clawed crayfish, and suggest a novel evolutive and phylogenetic scenario for A. pallipes populations of Southern Europe.
Table 1 Systematic and taxonomic revisions of Austropotamobius pallipes complex (from Grandjean et al. (2000) modified). Authors
Genus
Species
Subspecies
Data
Lereboullet (1858) Bott (1950, 1972)
Austropotamobius Austropotamobius
pallipes pallipes
pallipes pallipes italicus lusitanicus
Morphology Morphology
Karaman (1963)
Austropotamobius
berndhauseri pallipes italicus
Albrecht (1982)
Austropotamobius
pallipes
Brodsky (1983)
Austropotamobius
pallipes italicus
Pretzmann (1987)
Austropotamobius
Starobogatov (1995)
Atlantoastacus
pallipes pallipes pallipes fulcisianus
orientalis lusitanicus pallipes italicus pallipes
Santucci et al. (1997)
Austropotamobius
Grandjean et al. (1998)
Austropotamobius
Grandjean et al. (2000)
Austropotamobius
Grandjean et al. (2002)
Austropotamobius
Fratini et al. (2005)
Austropotamobius
pallipes italicus
Trontelj et al. (2005)
Austropotamobius
pallipes
pallipes italicus
berndhauseri? pallipes italicus
Morphology italicus lusitanicus carsicus pallipes lombardicus carinthiacus trentinicus pallipes bispinosus italicus lusitanicus carsicus pallipes fulcisianus = italicus pallipes rhodanicus fulcisianus = berndhauseri + A. p. lombardicus + A. p. trentinicus italicus orientalis = carsicus carinthiacus
Morphology
Morphology
Morphology Morphology
Allozyme pallipes italicus lusitanicus pallipes italicus carsicus carinthiacus Not sampled pallipes italicus carsicus carinthiacus pallipes italicus meridionalis carsicus carinthiacus pallipes
mtDNA RFLP Morphology 16S rDNA
16S rDNA
16S rDNA
COI mtDNA
S. Chiesa et al. / Molecular Phylogenetics and Evolution 61 (2011) 1–11
2. Materials and methods 2.1. Collecting samples In total, 356 white-clawed crayfish were collected by hand and nets in 38 different sampling sites distributed from the Alps to the most southern peninsular areas of Italy, a ‘‘hotspot’’ for the diversity of the species (Souty Grosset et al., 2006). From each individual, a portion of muscle tissue was collected from fragments of pleiopods and chelae taken from live samples, avoiding the death of the animals. Crayfish were released alive in the same collection site after tissue sampling. Genomic DNA was extracted from all samples but to ensure high reliability of the data, only 199 samples from 35 populations having high quality DNA were retained for molecular analysis (Fig. 1, Table 2). In the next sections the sampled populations will be labeled with alpha numerical codes based on the 16S mtDNA haplotype (see Grandjean et al., 2002; Fratini et al., 2005) and sampling location following a northern–southern gradient. Populations previously characterized by 16S mtDNA (Grandjean et al., 2002; Fratini et al., 2005) were now submitted to COI haplotyping and AFLP fingerprinting. COI haplogroups acronyms used reflect those proposed by Trontelj et al. (2005). In addition, three individuals of Austropotamobius torrentium (Schrank, 1803) from the Magyar Termeszettudomanyi Muzeum of Budapest (Hungary) were included as out-group for both the mitochondrial and AFLP analysis. All tissue samples are actually
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conserved in absolute ethanol at 20 °C, at the Laboratory of Molecular Zoology, Department of Evolutionary and Functional Biology, University of Parma, Parma (Italy). 2.2. DNA extraction and purification High molecular weight genomic DNAs were extracted and purified from ethanol-fixed muscle tissue stored at 20 °C. DNA was extracted either according to the classical SDS-proteinase K and phenol–chloroform technique described by Moore (1999) or alternatively by Aquapure genomic DNA kit (Biorad). DNA quality was visually inspected by 1% agarose gel electrophoresis in TAE buffer. All DNA samples are actually conserved in rehydration solution at 20 °C, at the Laboratory of Molecular Zoology, Department of Evolutionary and Functional Biology, University of Parma, Parma (Italy). 2.3. Sequencing of mitochondrial DNA COI gene fragment was amplified using the primers HCO 2198 and LCO 1490 (Folmer et al., 1994; Trontelj et al., 2005; Dawnay et al., 2007), which presumably should avoid the problem of pseudogenes detection, since they have been extensively used in many invertebrate and vertebrate species. A reaction volume of 25 ll containing 1U di GoTaq (Promega), Mg2+ 2.5 mM and dNTPs 0.2 mM was used. PCR profile was set as follows: 40 cycles of
Fig. 1. Sampling sites of the A. pallipes populations analysed. The different A. pallipes populations labeled with alpha numerical codes based on the 16S mtDNA haplotype (see Grandjean et al., 2002; Fratini et al., 2005), and sampling location following a northern–southern gradient.
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S. Chiesa et al. / Molecular Phylogenetics and Evolution 61 (2011) 1–11 Table 2 Population identification number, sampling sites, 16S haplotype and number of analysed specimen of the A. pallipes and A. torrentium, submitted to COI characterization (bold) and AFLP fingerprinting. CS = 16S carsicus haplotype; CR = 16S carinthiacus haplotype, P = 16S pallipes haplotype, I = 16S italicus haplotype; M = 16S meridionalis haplotype (haplotypes according to Grandjean et al. (2002) and Fratini et al. (2005)). No.
Sampling sites
16S Haplotype
No. individuals AFLP
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
Udine Rio Valturcana (Belluno) Roggia di Terlago (Trento) Rio Gamberi (Verbania) Risorgive del Ticino (Novara) Fontanile (Cremona) Bragamonti (Cuneo) Valle Peso (Cuneo) Arandolo (Cuneo) Torrente Rossana (Cuneo) Torrente Oxentina (Imperia) Rio Rialasco (Genova) Torrente Nenno (Genova) Torrente Chiusola (La Spezia) Torrente Stirone (Parma) Lago Pranda (Reggio Emilia) Torrente Zena (Bologna) Pistoia Rio Canvella (Prato) Il Mulino (Firenze) Castagno d’Andrea (Firenze) Rio Ceppeta (Arezzo) Fosso Doglio (Perugia) Fosso Argentina (Terni) Fosso Olpeta (Viterbo) Fosso Ariana (Rieti) Fosso dei Pantani (Rieti) Torrente Licenza (Roma) Fosso Duranna (Roma) Chieti Torrente Verde (Chieti) Fosso Vetrina (Chieti) Bussento (Salerno) Bussentino (Salerno) Villa D’Agri (Potenza) Magyar Termeszettudomanyi Muzeum of Budapest
CS1 CS2 CS3 CR4 CR5 CR6 P7 P8 P9 P10 P11 P12 P13 CR14 CR15 CR16 I17 I18 I19 I20 I21 I22 I23 M24 M25 M26 M27 M28 M29 M30 M31 M32 M33 M34 M35 A. torrentium
7 20 5 4 4 5 3 3 5 7 3 3 10 2 4 5 2 10 7 1 11 14 5 2 3 4 4 12 3 2 3 3 4 7 12 3
30 s at 95 °C, 45 s at 45 °C, and 1 min at 72 °C, after an initial 10 min denaturation step at 95 °C and a final extension at 72 °C for 10 min. PCR products were purified by elution from a 2.5% agarose gel, then precipitated with three volumes of 100% ethanol, and washed with 70% ethanol. Fragments sequencing was performed by CEQ™ DTCS-Quick Start Kit (Beckman Coulter) and analyzed on ‘‘CEQ™ 8000 DNA Analysis System’’ (Beckman Coulter). The amplified fragments consistently reached about 650 bp length. They were compared with sequences available in genomic databases using Blast and multiple alignments of sense and antisense sequences were conducted using such softwares as Clustal X (Thompson et al., 1997) and Sequencer 4.2 (Gene Code Corporation), verifying the correctness of the alignment also at the amino acid level. 2.4. Production and scoring of fluorescent AFLP markers AFLP markers were produced following the protocol already validated by Papa et al. (2005) and Maldini et al. (2006). Compared to the original protocol by Vos et al. (1995), herein the extract and purified genomic DNAs were cut by TaqI/EcoRI endonucleases, which showed higher polymorphism in animal genomes. Five primer combinations were used (in brackets the selective nucleotides): E32-T33 (eAAC-tAAG); E33-T32 (eAAG-tAAC); E32-T32 (eAAC-tAAC); E40-T37 (eAGC-tACG); E33-T37 (eAAG-tACG). An amount of 1.7 ll of PCR product and 0.3 ll of DNA internal size standards (CEQ DNA Size standard-600 Beckman-Coulter, Fullerton, CA) were added to 40 ll of deionized formamide (J.T. Baker, Phillipsburg, NJ). Samples were then loaded into the ‘‘CEQ™ 8000
No. individuals COI mtDNA 2 3 2 4
10 1
8
3
1 1
DNA Analysis System’’ (Beckman Coulter). Running conditions for capillary electrophoresis, raw data elaboration and reproducibility was tested according to Papa et al. (2005). A minimum fluorescence threshold value of 2000 was chosen on the basis of several comparative analyses, but lower peaks have sometimes been considered, especially for high molecular weight fragments when their resolution was comparable to those of ladder fragments having similar size. Fragments useful to assess genetic differentiation within species were identified as those reaching the threshold peak and being well distinct from nearby peaks (Papa et al., 2005). Analysed data was imported in Genographer software (Vers.1.6.0, Benham J.J., Montana State University 2001), for band scoring. Genographer allowed the construction of a virtual gel with bands shaped on the base of peak height, resolution and mobility and a thorough analysis of single fragments. Fragments in each individual were binary scored as 1 or 0, following the ‘‘band-based’’ approach (Bonin et al., 2007). To avoid genotyping errors in the AFLP procedure all the recommendations by Pompanon et al. (2005) and Bonin et al. (2007) were accomplished: (1) high quality DNA (2) standardized and validated procedures (3) replication of 30% of the samples, coupled with systematic duplication of samples (4) reliability of the results checked among different laboratories (5) double independent reading of the profiles carried out by researchers. 2.5. Phylogenetic and statistical analysis The Median Joining Network of COI gene sequences was built using Network 4.02-05 (http://www.fluxus-technology.com)
S. Chiesa et al. / Molecular Phylogenetics and Evolution 61 (2011) 1–11
according to Bandelt et al. (1999). It has been chosen to include missing haplotypes by generating median vectors. Moreover, it has been tested by Cassens et al. (2005) and Mardulyn et al. (2009) to be one of the most appropriate methods to infer genealogical relationships among intraspecific sequences, as one of the methods generating the smallest number of errors. The evolutionary tree of DNA sequences has been estimated by the maximum likelihood optimal criterion developed by Felsenstein and Churchill (1996). Over the traditional parsimony algorithms, which can give misleading results if rates of evolution differ in different lineages, the ML model assumes that each lineages and each site evolves independently and it allows the expected frequencies of the four bases to be unequal. The confidence level of the phylogenetic tree has been assessed by the bootstrapping technique proposed by Felsenstein (1985). The confidence for each clade of the observed tree has been estimated on the proportion of 1000 bootstrap trees showing that same clade. Finally, Factorial Correspondence Analysis of AFLP markers was undertaken by Genetix software (Belkhir et al., 1996–2002) where correspondences are adapted to the diploid genotypes and can be depicted graphically in 3D if desired. The FCA analyzed objects (group of individuals) are seen like a group of dots in a hyperspace which has as many dimensions as terms (alleles-bands) for all the variables (alleles-bands at different loci). The algorithm seeks the independent directions (orthogonal) in this hyperspace the length whose inertia – size which, by analogy with physics, represents the integral of the mass (here e.g. the number of individuals in a point of the hyperspace) multiplied by the square of the distance to the
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center of the co-ordinates (here called center of gravity) – is maximized. These directions, which are defined by the appropriate vectors of the matrix (C–D from linear combinations of the original vector variables), determine a series of factorial axes. By convention, the first axis is that which has the strongest total contribution to the inertia. With AFLP bands, to use the individual genotypic data each individual is represented by his score for each term of each variable, that is to say 0 for the absence, 1 for the presence of the AFLP band. For each axis determined at the time of the analysis, a set of coefficients for each individual and of the locus are also calculated, as a matter of: 1. Of the absolute contributions: expressing the share taken by an element given (individual or alleles) in the inertia explained by a factor. 2. Of the relative contributions: expresses the share taken by the axis in the total contribution of the individual or the allele to the inertia (representing the dispersion of the cloud of the points). 3. Of the co-ordinates of all the points individuals and alleles with the various axes, safeguarded in a file. This last can be used in another graphic utility to draw groups of dots. These co-ordinates are also used to draw groups of dots, into two or three dimensions, to which it is possible to make undergo rotations and zooms, to visualize them under different angles. 3. Results 3.1. COI mitochondrial DNA Thirty-three samples belonging to nine populations representative of the geographic sampling area were selected for COI gene
Fig. 2. Median Joining Network of COI gene sequences. Median Joining Network obtained from the COI gene sequences with mutational steps numbered. For example, P13_1 means P haplotype group, population 13, specimen 1. See Table 1 for details. WEURO: Western Europe haplotype group (Trontelj et al., 2005); NWITAL: North Western Italy haplotype group (Trontelj et al., 2005); APPEN: Apennine haplotype group (Trontelj et al., 2005); SEA-WB: South Eastern Alps–Western Balkans haplotype group (Trontelj et al., 2005); ISTRIA: Istria haplotype group (Trontelj et al., 2005); CASER: Caserta sequences from Southern Apennines (Aceto et al., 2008).
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Fig. 3. Maximum Likelihood tree inferred by the COI gene sequences analysis. For example, P13 1 means P haplotype, population 13, specimen 1. See Table 1 for details.
analysis (Table 2). We amplified and sequenced 650 bp of the gene, while the alignments were based on 399 bp GenBank reference sequences (Supplementary Table, Supporting Information for online publication only). A range of 5–25 mutational steps were detected among the aligned sequences of different haplotype groups of white-clawed crayfish, with limited or null transversions
(Table 3). The total number of nucleotide substitutions between the A. pallipes and the outgroup A. torrentium ranged between 45 and 52 (considering both transitions and transversions) (Table 3). A detailed description of nucleotide diversity is reported in Table 3. Median Joining Network revealed a strong geographic structure with at least four distinct haplogroups separated by a variable
Table 3 Total number of nucleotide substitutions between A. pallipes and the outgroup A. torrentium, and among A. pallipes lineages (Tr = Transitions; Tv = Transversions). WEurope = Western Europe haplotype group; NWItaly = North Western Italy haplotype group; SEAlps-WBalkans = South Eastern Alps–Western Balkans haplotype group; Apennine = Apennine haplotype group (geographical abbreviations according to Trontelj et al. (2005)). Sequences of A. pallipes were grouped to detect haplogroups on the basis of highly diagnostic mutations.
A. torrentium WEurope NWItaly SEAlps-WBalkans Apennine
A. torrentium
WEurope
NWItaly
SEAlps-WBalkans
Apennine
– 52 48 45 48
52 – 23 25 23
48 23 – 11 12
45 (30 Tr-15 Tv) 25 (21 Tr-4 Tv) 11 (11 Tr-0 Tv) – 5 (5 Tr-0 Tv)
48 (30 Tr18 Tv) 23 (18 Tr-5 Tv) 12 (12 Tr-0 Tv) 5 (5 Tr-0 Tv) –
(33 (30 (30 (30
Tr-19 Tr-18 Tr-15 Tr-18
Tv) Tv) Tv) Tv)
(33 Tr-19 Tv) (18 Tr-5 Tv) (21 Tr4 Tv) (18 Tr-5 Tv)
(30 Tr-18 Tv) (18-5 Tv) (11 Tr-0 Tv) (12 Tr-0 Tv)
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number of mutations (Fig. 2). Accordingly, Maximum Likelihood tree revealed four groups supported by bootstrap values ranging between 650 and 1000 (Fig. 3) and consistent with the haplogroups previously described by Trontelj et al. (2005). No match with the additional Istria reference sequence was identified. The Istrian haplotype identified by Trontelj et al. (2005) was not found and therefore omitted in Table 3. In detail, the populations from Northern and Central Italy (CR4, CR5, CR15 and I18) grouped with the ‘‘North Western Italy’’ haplotype group, far apart from sequences of the population of Western Italy P13, which were grouped in the haplotype group of ‘‘Western Europe’’ (seven polymorphic sites). The sequences from populations M28 and M35 of Central and Southern Italy, clustered together with the ‘‘Apennine’’ haplotype group; while the specimen from North Eastern Italy, belonging to the populations CS1 and CS2, were assigned to the ‘‘South Eastern Alps–Western Balkans’’ haplotype group. Genetic variability within haplogroups ranged between 7 and 32 point mutations considering both our samples and reference sequences while within the populations analysed in this work the number was comprised between 5 and 10.
3.2. AFLP markers A total of 1010 AFLP reliable fingerprinting profiles were obtained genotyping 202 samples (199 A. pallipes and 3 A. torrentium) with 5 EcoRI/TaqI primer combinations carrying three selective nucleotides (Fig. 4). The repeatability was greater than 99%. A total of 535 AFLP bands in a range 50–500 bp, 479 of which polymorphic (89%) and only 56 monomorphic across the dataset, were binary scored. The mean number of bands per primer combination was 107; the monomorphic bands ranged from 8 to 15, while the polymorphic ones ranged from 86 to 107 depending on the primer pairs (Table 4).
Fig. 4. Genographer virtual gel with AFLP profiles. Fingerprinting profiles of single crayfish (columns) and AFLP loci (rows) detected by Genographer software.
The expected heterozygosis calculated within populations ranged from 0.07 (S.d. = 0.02) in M24 to 0.31 (S.d. = 0.01) in M35, with a mean value of 0.17 (S.d. = 0.01). The Gst indexes were 0.48 among the whole dataset (outgroup included) and 0.46 among A. pallipes populations indicating an overall genetic differentiation between different populations. The Nei’s distances between A. pallipes populations and A. torrentium ranged from 0.27 (CR15 vs A. torrentium) to 0.59 (P13 vs torrentium) while within the pallipes populations the distances ranged between 0.06 (CR5 vs I17) and 0.32 (P12 vs M33). Focusing on samples belonging to the same COI haplotype group, Nei’s genetic distances based on AFLP polymorphisms were in the range 0.15–0.38 for N Western Italy (respectively I18 vs CR4 and I18 vs CR15), 0.16 for Apennine (M28 vs M35), 0.14 for S Eastern Alps–Western Balkans (CS1 vs CS2) . The Factorial Correspondence Analysis (FCA) at individual level (Fig. 5) showed a clear separation between A. torrentium and A. pallipes only. Indeed, no clear clusters gathering populations of A. pallipes were detected among the 35 analyzed groups. The inertia explained by the first three axes were 15.53%; 10.51% and 8.68% respectively. In spite of the mitochondrial divergence, the nuclear genome showed a weak differentiation. For example, individuals belonging to I18 and P13 described as different species by Grandjean et al. (2002) on the basis of mitochondrial 16S lineages, showed COI mtDNA separated by 23 nucleotide mutational steps, but they shared 144 monomorphic AFLP bands out of 535 (27%). Only 42 bands out of 535 (9%) were diagnostic (fixed in a group and absent in the other).
4. Discussion Until now, sequencing of mitochondrial gene fragments has been the most common method to study crayfish phylogeny, either for the genus Austropotamobius, Euastacus (Clark, 1936) (Shull et al., 2005), Orconectes (Cope, 1872) (Mathews et al., 2008), Cambaroides (Faxon, 1884) (Ahn et al., 2006; Braband et al., 2006), and Cherax (Erichson, 1846) (Munasinghe et al., 2004; Nguyen et al., 2004). Analyses of COI gene fragment of A. pallipes from widely distributed Italian populations identified four major haplogroups, overlapping those previously described by Trontelj et al. (2005) and Stefani et al. (2009). However, results were only partially matching the mitochondrial lineages described by Grandjean et al. (2002) and Fratini et al. (2005) with a different marker. More precisely, CR (carinthiacus) and I (italicus) lineages, considered distinct subspecies by the 16S mtDNA (Grandjean et al., 2002; Fratini et al., 2005), in this work resulted in the same COI haplogroup defined ‘‘North Western Italy’’ (assimilated to previously defined italicus lineage). As a consequence, the hypothesis of two different subspecies, already considered doubtful (Machino, 1997; Trontelj et al., 2005), has also been heavily challenged by our results.
Table 4 Number of monomorphic, polymorphic, total loci, and percentage of polymorphism scored by AFLP, totally and for each primer combination. Primer combination
Monomorphic loci
Polymorphic loci
Total
% Polymorphism
eAGC tACG eAAG tACG eAAG tAAC eAAC tAAC eAAC tAAG Total 5
12 9 8 15 12 56
86 104 86 96 107 479
98 113 94 111 119 535
88 92 91 86 90 90
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Fig. 5. Two-dimensional FCA of the 202 analysed individuals generated by Genetix software. Individuals were grouped to detect putative species A. pallipes–A. italicus (sensu Grandjean et al., 2002; Fratini et al., 2005) genotyped by AFLP. Squares: A. pallipes; Triangles: A. italicus; Circles: A. torrentium.
The median-joining network showed both a differentiation between lineages and a clear geographic pattern reflecting local genetic variation. Each population is characterized by a single haplotype, even if consistent mutational steps could be identified among sequences of the same population. However, incongruence related to the effectiveness in species recognition by mitochondrial sequences have been demonstrated in the recent years (Song et al., 2008; Galtier et al., 2009), mainly due to the identification of nuclear mitochondrial pseudogenes in crustacean genomes (Schneider-Broussard and Neigel, 1997; Bucklin et al., 1999; Bensasson et al., 2001; Williams and Knowlton, 2001; Nguyen et al., 2002; Song et al., 2008; Galtier et al., 2009). Similarly, Galtier et al. (2009) showed that in such particular groups as arthropods, characterization of mtDNA for taxonomy and systematics is not completely reliable due to the common hybrid introgression. Therefore, an integrated approach is potentially useful to investigate the composite relationships among species/subspecies and populations, and AFLPs revealed to be a valid class of nuclear markers to be coupled to mitochondrial analysis. As explained by Vernesi and Bruford (2009), conservation genetics has largely remained a field were a small number of molecular markers are applied to few populations of a single species, some of which may be threatened. The usefulness of AFLP markers for taxonomy and definition of closely related species has been largely proved in many zoological groups, as arthropods (Mendelson and Shaw, 2005; Ming and Wang, 2006). The AFLP markers possess features suitable for animal conservation genetics (Vernesi and Bruford, 2009): for surveys of intrapopulation variability and genetic structures among populations, AFLPs have proved to be effective as standards markers (Vernesi and Bruford, 2009). Furthermore, AFLPs can be successfully used for phylogenetics inferences (Sullivan et al., 2004). Flexibility, no need for
extensive technique development, and low cost make AFLPs potentially effective for conservation genetics studies (Lucchini, 2003). The limited use of AFLPs in animal conservation genetics seems even more paradoxical (Vernesi and Bruford, 2009). The percentage of polymorphic AFLPs (90%) in crayfish agreed with those reported in several studies on crustacean and insects, ranging from 65% to 95% (Fezner and Crandall, 1999; Mendelson and Shaw, 2002; Salvato et al., 2002; Cannas et al., 2003; Gili et al., 2004; Mendelson et al., 2004; Sun et al., 1999; Shuster et al., 2005), up to 99% in the Ortoptera Locusta migratoria manilensis (Meyen, 1835) (Zheng et al., 2006). Only few AFLP markers among five hundred fragments were highly diagnostic, and produced an unexpected limited overall differentiation between individuals and lineages. Only 9% of polymorphic bands were highly diagnostic between PAL (pallipes) and ITA (italicus) lineages, while in sibling species like Helicoverpa armigera (Hübner, 1805) and Helicoverpa assulta (Guenèe, 1852) (Lepidoptera) the percentage of species-specific bands was at least threefold higher (about 30% Ming and Wang, 2006). Despite the differentiated mitochondrial haplotype groups, AFLP fingerprinting indicated that a consistent part of the genome is shared among distant populations of A. pallipes. In particular, individuals belonging to the two putative species A. pallipes (P) and A. italicus (I) were clearly overlapping. AFLPs on A. pallipes therefore open a new perspective on its evolutionary history, suggesting the existence of a unique species in Italy. In similar cases AFLPs solved disputes regarding the taxonomy of putative species and subspecies of crustaceans, such as the ones belonging to genus Eriocheir (De Haan, 1835) (Lu et al., 2000) and Artemia (Leach, 1819) (Tryantaphyllidis et al., 1997), and revealed that the common pearl oysters Pinctada fucata (Gould, 1850) is monospecific (Yu et al., 2006).
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Incongruence between mitochondrial and nuclear signals were already reported in the phylogeny of Arthropods (Gomez-Zurita and Vogler, 2003), and in other taxa when comparing mtDNA sequencing with polymorphic markers as AFLPs (Egger et al., 2007; Koblmüller et al., 2007). The controversy is not only limited to our work, but it reflects the contradiction of DNA evolution in such groups, as the freshwater decapods, where mitochondrial and nuclear DNA could show different evolutionary histories. So how can we explain this complex scenario? The distribution of mitochondrial haplotype groups allowed Trontelj et al. (2005) to pinpoint the possible glacial refugia, such as microrefugia in Southern France (Alpes-Maritimes), NorthWestern Italy (Liguria), Apennine and Istrian region, the latter considered the primary center of radiation of A. pallipes. After the split between A. torrentium and A. pallipes in the Miocene (8–12 MYA); the separation of the basal mitochondrial lineages of A. pallipes (Western Europe, North Western Italy, South Eastern Alps– Western Balkans) occurred during the Pliocene (3–6 MYA), while the split of the Apennine lineage occurred during the Pleistocenic glaciations (0.1–1.5 MYA) (Trontelj et al., 2005). Despite different methodological approaches, our paper is in agreement with Santucci et al. (1997) that reached the following conclusion: ‘‘a wide genetic heterogeneity that appears mainly related to range fragmentation and subsequent recolonizations from multiple refugia during the glacial events’’. Such a pattern indicates that, beyond a general expansionspeciation model (Hewitt, 1996), the white-clawed crayfish experienced a more complex evolutionary history. This consideration was driven by the existence of several microrefugia for other freshwater invertebrates (Pongratz et al., 2003; Verovnik et al., 2004) and fish such as Thymallus thymallus (Linnaeaus,1758) (Weiss et al., 2002), Cottus gobio (Linnaeaus, 1758) (Englbrecht et al., 2000), and Leuciscus (Cuvier, 1816) (sin. Squalius Berg, 1949) species complex (Costedoat et al., 2006). The Balkan microrefugia generated the ‘‘South Eastern Alps–Western Balkans’’ (carsicus) lineage while the Southern France (Costedoat et al., 2006), and Alps Maritimes (Trontelj et al., 2005; Stefani et al., 2011) microrefugia differentiated the ‘‘Western Europe’’ (pallipes) and the ‘‘North Western Italy’’ (italicus + carinthiacus) lineages respectively. Also Central and Southern Apennines have been considered important refugial areas for Italian fauna (Trontelj et al., 2005), from which the ‘‘Apennine’’ (meridionalis) lineage emerged. Interestingly, Canestrelli et al. (2006) demonstrated that similar evolutionary events in amphibians were driven by fragmentation of the Apennine chain as separated islands during plio-pleistocene periods. To summarize, the white-clawed crayfish likely originated in the headwaters of the Eastern Alps–Western Balkans, migrated westward onto the Apuane Alps, and then proceeded southward through the Apennine in the Central and Southern Italy. During the Pleistocene, the alteration of glacial and interglacial phases caused the sea level variations that resulted in a minimum Mediterranean level 100–130 m below the present day level (Pielou, 1979). This process produced the emergence of the upper and middle Adriatic and Tyrrhenian continental shelves, favoring the river confluence of downstream courses (Bianco, 1990). In particular, the ancient location of the Po river delta in the Central-Southern Adriatic area (Southern Italy) permitted the direct connection between Italian and Croatian rivers. This event might have enabled several crayfish settling between the West and East Adriatic coasts, supporting the Eastern Adriatic regions/ Balkans as the center of original radiation (Trontelj et al., 2005). Bottleneck and genetic drift have then played a major role in shaping the present-day distribution of the mitochondrial haplotype groups. In fact, the number of median vectors connecting
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the network, suggests the existence of several intermediate unsampled lineages, likely already extinct. The distribution of the haplotypes might be also affected by the dispersal capability and gene flow of A. pallipes – a k-selected species – nowadays limited by habitat fragmentation, and severe demographic fluctuations, while in ancient times were enhanced by consistent river connection and better environmental quality. Finally, artificial propagations such as the well known translocation to the Iberian Peninsula from Northwestern Italy (Grandjean et al., 2002; Trontelj et al., 2005) and the introduction of crayfish from the Apennine group to Southern France and Northwestern Italy (Zaccara et al., 2004) probably had a massive influence on the distribution of A. pallipes in recent years. In conclusion, the analysis of COI gene sequences revealed a zoogeographic pattern not detectable in the nuclear genome of A. pallipes. In our view, AFLP fingerprinting witnesses a secondary intensive contact among the different phylogeographic lineages occurred during the postglacial events, due to the changes of habitats and freshwater drainages. This scenario was proposed also in other groups where nuclear genomic variation was not correlated withmtDNA differentiation (Koblmüller et al., 2007; Yang and Kenagy, 2009). In particular, Egger et al. (2007) found significant incongruence between phylogenetic reconstructions from mitochondrial DNA and AFLP data, suggesting incomplete sorting of mitochondrial haplotypes as well as frequent introgression between differentiated lineages of cichlid fishes. Hence, in absence of complete reproductive barriers, hybridization of nuclear genomes has been shown to be possible, and sister taxa still hybridize despite millions of years of divergence demonstrated by molecular data (Hewitt, 2000). Therefore, considering experimental molecular data and the above cited ecological considerations, the previously proposed haplotypes of A. pallipes are clearly nested within a unique taxon and do not support the existence of two sister species A. pallipes and A. italicus. Thus, we hereby named all the white-clawed crayfish populations as A. pallipes being the recognized species name due to priority. Acknowledgments This research was supported by the Italian Ministry of University and Research, as a Ph.D. Project at the University of ‘‘Roma Tre’’, Rome (Italy). The authors would like to thank people and Authorities for their help with collecting samples of A. pallipes: G. Caricato (Basilicata Region), M. De Biaggi (Piedmont), G. De Luise (Friuli Venezia-Giulia), F. Gherardi and S. Fratini (Tuscany), T. Pagliani (Abruzzo and Marche), E. Marconato (Veneto and Lombardia), S. Salvidio (Liguria), M. Zanetti (Veneto) and Lazlo Forrò from the Magyar Termeszettudomanyi Muzeum of Budapest (Hungary) for A. torrentium samples. Authors would like to thank anonymous referees for improving suggestions and also thank Dr. Kristin Brabender for English language corrections. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ympev.2011.03.031. References Aceto, S., De Vico, G., Sica, M., Maio, N., Carella, F., Cataldi, M., Canestrini, F., Froglia, C., 2008. Mitochondrial COI gene sequences of Austropotamobius pallipes complex from Reggia di Caserta (Italy). Direct Submission. Available from: www.ncbi.nlm.nih.gov.
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