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. 2011 Jun 28;108(2):241–252. doi: 10.1093/aob/mcr134

The importance of Anatolian mountains as the cradle of global diversity in Arabis alpina, a key arctic–alpine species

Stephen W Ansell 1,*, Hans K Stenøien 3, Michael Grundmann 1, Stephen J Russell 2, Marcus A Koch 4, Harald Schneider 1, Johannes C Vogel 1
PMCID: PMC3143044  PMID: 21712298

Abstract

Background and Aims

Anatolia is a biologically diverse, but phylogeographically under-explored region. It is described as either a centre of origin and long-term Pleistocene refugium, or as a centre for genetic amalgamation, fed from distinct neighbouring refugia. These contrasting hypotheses are tested through a global phylogeographic analysis of the arctic–alpine herb, Arabis alpina.

Methods

Herbarium and field collections were used to sample comprehensively the entire global range, with special focus on Anatolia and Levant. Sequence variation in the chloroplast DNA trnL-trnF region was examined in 483 accessions. A haplotype genealogy was constructed and phylogeographic methods, demographic analysis and divergence time estimations were used to identify the centres of diversity and to infer colonization history.

Key Results

Fifty-seven haplotypes were recovered, belonging to three haplogroups with non-overlapping distributions in (1) North America/Europe/northern Africa, (2) the Caucuses/Iranian Plateau/Arabian Peninsula and (3) Ethiopia–eastern Africa. All haplogroups occur within Anatolia, and all intermediate haplotypes linking the three haplogroups are endemic to central Anatolia and Levant, where haplotypic and nucleotide diversities exceeded all other regions. The local pattern of haplotype distribution strongly resembles the global pattern, and the haplotypes began to diverge approx. 2·7 Mya, coinciding with the climate cooling of the early Middle Pleistocene.

Conclusions

The phylogeographic structure of Arabis alpina is consistent with Anatolia being the cradle of origin for global genetic diversification. The highly structured landscape in combination with the Pleistocene climate fluctuations has created a network of mountain refugia and the accumulation of spatially arranged genotypes. This local Pleistocene population history has subsequently left a genetic imprint at the global scale, through four range expansions from the Anatolian diversity centre into Europe, the Near East, Arabia and Africa. Hence this study also illustrates the importance of sampling and scaling effects when translating global from local diversity patterns during phylogeographic analyses.

Keywords: Anatolia, centre of origin, Pleistocene glaciations, chloroplast trnL-F, divergence times, alpine plants, Arabis alpina

INTRODUCTION

Genetic diversity is often structured within species distribution ranges (Taberlet, 1998; Hewitt, 2000, 2004; Médail and Diadema, 2009). Local hotspots of diversity may arise through several means: the long-term survival of populations during past climatic fluctuations (glacial refugia; Hewitt, 2004; Schmitt, 2007), or through the recent amalgamation of divergent lineages (Ferris et al., 1998; Petit et al., 2003; Walter and Epperson, 2005), or local ecological niche gradients may promote the accumulation of lineages (Ricklefs, 2007). Discriminating among the first two scenarios still remains a challenge when interpreting phylogeographic history, particularly outside the much studied European and North American continents.

Anatolia is a hotspot for biological diversity in the Mediterranean basin and is a biogeographically interesting, but under-explored region. It is situated between the European and Asian continents and is a meeting point for the European and Turko-Iranian floras, which broadly overlap in central Anatolia (Davis, 1965–85). During the Pleistocene (approx. 2·5 Mya) glacier development within Anatolia was limited to the higher mountain peaks (Atalay, 1996), while the lowlands remained open, developing steppe communities (Michaux et al., 2004; Magyari et al., 2008). These provided suitable habitats for temperate species to survive the last glacial maximum (Rokas et al., 2003; Dubey et al., 2006; Fritz et al., 2009) and perhaps in earlier cycles. Late Pleistocene sea levels around the Mediterranean and Caspian seas were regularly lower than current levels (approx. 130 m, Kerey et al., 2004) and land bridges routinely formed across the Sea of Marmara and the Bosporus Strait (Magyari et al., 2008). This allowed for frequent inter-continental population exchange, especially for temperate organisms (Kučera et al., 2006; Dubey et al., 2007; Gündüz et al., 2007). In the east, the high ‘Anatolia diagonal’ (Davis, 1971) mountains (>2000 m) have limited the mixing of European and Near-Eastern biota (Ekim and Güner, 1986; Bilgin et al., 2006), but their topography may also have facilitated local survival and speciation, as suggested by the high level of endemism (Davis, 1971; Çiplak, 2003). Together this combination of geography and climatic history has promoted both the long-term local survival of biota, and repeated uni- or bi-directional colonization(s) from nearby areas, making Anatolia a complex but important area for understanding the diversification of Eurasian biodiversity.

Increasingly the biogeographic history of Anatolia and adjoining areas is being explored using molecular tools. While data are still limited, bats, rodent, turtles and wasp groups are now represented (e.g. Michaux et al., 2004; Bilgin et al., 2006; Dubey et al., 2007; Gündüz et al., 2007; Flanders et al., 2009; Furman et al., 2009; Fritz et al., 2009). Plants remain relatively poorly represented, despite several studies focusing on trees (Bartish et al., 2006; Kučera et al., 2006; Gömöry et al., 2007; Naydenov et al., 2007) or herbs (Jakob et al., 2007; Font et al., 2009; Parolly et al., 2010). Unfortunately low sampling densities have limited the ability to characterize the location of refugia, hybrid zones and colonization routes (Gündüz et al., 2007). Nevertheless several phylogeographic trends emerge from these studies, but with no clear consensus across all groups: (1) Near East and European lineages are genetically divergent; (2) Anatolia was the source area for European colonization; (3) the Thracian Plain/Sea of Marmara is a barrier to European colonization; and (4) the high Anatolian diagonal mountains are a barrier to colonization from the Near East (Fig. 1). Phylogeographic information from alpine species is currently missing and would complement the existing information from temperate species, especially in a context of the Anatolian mountains as a refugium.

Fig. 1.

Fig. 1.

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Some important Anatolian geographic features. Diagonal adapted from Davis (1971).

The arctic–alpine herb Arabis alpina was selected to explore the phylogeographic history of the Anatolia mountains and relationships with adjacent mountainous areas. Arabis alpina is a short-lived perennial belonging to the mustard family Brassicaceae (Hegi, 1986), and a relative of the model organism Arabidopsis thaliana (Koch et al., 2000, 2001, 2007; Beilstein et al., 2008; Couvreur et al., 2010). Arabis alpina itself is currently being developed as a model for studying alpine and life-history adaptation (Wang et al., 2009; Poncet et al., 2010), although complicated by loss of self-incompatibility in parts of the Europe (Tedder et al., 2011). The herb is distributed throughout the alpine habitats in Europe, in the arctic zone of Greenland and North America, in the high mountains of northern and eastern Africa and Anatolia; and in the ranges extending into the Caucasus and the Near East (Meusel et al., 1965). The global phylogeographic structure of A. alpina was outlined by an earlier chloroplast DNA studies, and three haplogroups were recovered from the core areas adjacent to Anatolia: Arctic/Europe/north Africa, the Near East and east Africa (Koch et al., 2006; Assefa et al., 2007; Ehrich et al., 2007). The importance of Anatolia at the centre of the global diversity range for A. alpina was recognized by a ‘three-times out of Asia-Minor’ hypothesis (Koch et al., 2006), but sampling was modest (n = 3). An alternative hypothesis of Anatolia as a centre for the amalgamation of European and Near East lineages is considered for other Brassicaceae (Kučera et al., 2006). These two phylogeographic hypotheses depend on the availability of suitable ecological and geographical connections between Anatolia and adjoining territories during the Pleistocene. This is especially true for alpine species like A. alpina, as colonizations between Anatolian and adjoining mountain systems are limited to glacial periods when alpine habitats are more widespread at lower elevations. As such we recognize that two key geographic features may have hindered or aided Pleistocene colonization of Anatolia: the Thyracian plain and the ‘Anatolian diagonal’. Consequently we put forward two additional hypotheses: (1) that the low laying areas around the Sea of Marmara and adjacent Thyracian plain (<1000 m) with its Mediterranean climate have presented an ecological barrier to European/Asian migrations; (2) that the high mountains of the ‘Anatolian diagonal’ have allowed population exchange between Anatolia and adjacent Caucasus and Near East mountain systems during ecologically favourable glacial periods.

To explore these two alternative hypotheses the following specific questions are raised. (1) Is Anatolia a centre of global genetic diversity for A. alpina ? (2) Is the local Anatolian diversity comprised of related haplotypes, as expected under a scenario of in-situ survival and local lineage accumulation? (3) Is genetic diversity geographically partitioned around the Sea of Marmara? (4) Is there evidence of range expansion associated with the ‘Anatolian diagonal’. (5) In what way has the Anatolian diversity contributed to the diversity of Europe, the Near East and eastern Africa? These questions are addressed by a global chloroplast DNA phylogeographic analysis of A. alpina, using the herbarium collections from the Flora of Turkey project (Davis, 1965–1985) as a sampling resource to place special focus on Anatolia and associated Levant.

MATERIALS AND METHODS

Leaf material from 182 individuals of Arabis alpina L., representing 135 previously unpublished localities, were sampled from herbarium specimens deposited in the following herbaria; Berlin-Dahlem (B), Copenhagen (C), the Natural History Museum London (BM) and the Royal Botanical Gardens Edinburgh (E). This sampling targeted mainly Iran, Iraq, Lebanon, Saudi Arabia, Syria, Turkey and Yemen. To a lesser extent, material was sampled from Armenia, Azerbaijan, Crete, Crimea (Ukraine), Georgia, Greece, Ethiopia, Kenya, Tanzania, Uganda and former Yugoslavian states (Supplementary Data Table S1, available online). The GLOBAL GAZETTER version 2·1 (http://www.fallingrain.com/world) was used to georeference herbarium voucher localities with the derived latitude/longitude co-ordinates reduced to two decimal places.

Total genomic DNA was extracted from the leaf tissue using a combination of the Retsch Tissue Lyser and Biosprint 96 BioRobot (Qiagen) workstation and the Biosprint 96 plant DNA extraction Kit (Qiagen). The trnL-trnF (trnL-F) region including the trnL(UAA) gene and the trnL-trnF(GAA) intergenic spacer (IGS) was amplified using PCR and the ‘C’ and ‘F’ primers (Taberlet et al., 1991), according to the conditions specified in Ansell et al. (2007). Both DNA strands were sequenced using BigDye Terminator Kit version 1·3 (Applied Biosystems, ABI), and an ABI 3730 capillary DNA analyser. The trnL-F IGS sequences were assembled using SEQMAN version 6·00 (Lasergene, DNAstar).

Sequence data from published studies, especially for European and eastern African specimens were also utilized. The following chloroplast trnL-F IGS sequences of Arabis alpina were recovered from GenBank: DQ060112–DQ060145 (Koch et al., 2006; 142 accessions world-wide, including 122 from Europe, Greenland and Canada), EF449508-EF449514 (Assefa et al., 2007; 66 accessions from 11 populations in the eastern African mountains) and EU403083–EU403084 (Ansell et al., 2008; 105 accessions from 34 populations in the Italian mountains and European Alps). The total dataset comprised 483 trnL-F IGS sequences from 296 locations (Supplementary Data Table S1).

Data analysis

Sequences from the new and published studies were aligned using Clustal X algorithm (Thompson et al., 1994) and subsequently manually corrected using MEGALIGN version 6·00 (Lasergene, DNAstar). New haplotypes types were submitted to GenBank (accession numbers JF705219–705252). Alignment positions 419–435 comprised a highly variable microsatellite C(1–5)T(8–12) (Supplementary Data Table S2), which was excluded during haplotype discrimination. Two haplotypes previously reported as being private to single accessions from Croatia and Iraq (types 16 and 31; Koch et al., 2006) where reclassified as haplotypes 13 and 04 respectively after examination of the original electropherograms. In total, 57 unique trnL-F haplotypes are recognized for A. alpina. The relationship among haplotypes was reconstructed using statistical parsimony with a 95 % connection threshold (Templeton, 1992) and the TCS program version 1·21 (Clement et al., 2000). All haplotypes were re-classified to obtain a unifying scheme (Supplementary Data Table S2) to overcome the previous adoption of both Arabic numeric and Roman numeral haplotype naming schemes (Koch et al., 2006; Assefa et al., 2007).

Samples were organized into ten groupings based on mountain range and observed haplotype distributions (Fig. 2A and Supplementary Data Table S1). Haplotype richness, haplotype diversity and nucleotide diversity was estimated for each region.

Fig. 2.

Fig. 2.

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(A) Global distribution range of Arabis alpina, and the three major chloroplast trnL-trnF IGS haplotype groups. Distribution adapted from Meusel et al. (1965) and Jalas and Suominen (1994), with dotted lines delimiting the ten regions used for genetic analysis. (B) The genealogical relationship among the 57 unique chloroplast haplotypes, with inferred haplotype sequences shown in black. (C, D) Regional distributions of haplotypes in Asia Minor (C) and the Arabian Peninsula and Ethiopia (D). Locations with multiple samples are indicated by black dots and connecting lines define regions: (1) Arctic and North America, (2) western and central Europe, (3) north Africa and Macronesia, (4) south-east Europe, (5) western Anatolia, (6) central Anatolia and Levant, (7) Caucasus and Iranian Plateau, (8) Arabian Peninsula, (9) Ethiopia, (10) east Africa. n, Sample size.

Haplotype richness (R) with correction for the smallest regional sample size (n = 16) through rarefraction (El Mousadik and Petit, 1996) was calculated using CONTRIB (Petit et al., 1998). Nucleotide diversity (π; Nei, 1987) and genetic differentiation by analysis of molecular variance procedure (AMOVA) were calculated using ARLEQUIN version 3·10 (Excoffier et al., 2005). Differentiation was initially calculated from haplotype frequencies (FST), subsequently incorporating haplotype pairwise genetic distances (ΦST), as the number of character changes. The significance of differentiation was assessed by 1000 non-parametric permutation tests (Excoffier et al., 1992), as performed by ARLEQUIN. GST and NST are analogous to FST and ΦST (Pons and Petit, 1996), and when the genealogical relationship of haplotypes is significantly correlated to geographic distribution of haplotypes, estimates of NST (ΦST) are predicted to be significantly greater than GST (FST) (Pons and Petit, 1996). Estimates of GST and NST were calculated using PERMUT version 2·0 (Pons and Petit, 1996), and the significance of difference between the two was assessed by 1000 permutations.

Demographic history was explored by mismatch distribution analyses of observed haplotype pairwise differences, following a sudden population expansion model (Rogers and Harpending, 1992), employing 1000 bootstraps, as performed by ARLEQUIN. Prior to estimating among haplogroup divergence time, each haplogroup was tested for deviations from neutral expectations by Tajima's D test (Tajima, 1989), as implemented in DnaSP version 4·50·3 (Rozas et al., 2003), and intraspecific recombination was tested using the SITES software (Hey and Wakeley, 1997). Since no deviations from neutrality and no signs of intraspecific recombination were found, time since divergence between haplogroups was estimated by the IM program (Nielsen and Wakeley, 2001; Hey and Nielsen, 2004). IM implements a Bayesian analysis approach based on coalescent theory and using Markov Chain Monte Carlo methodology to sample from a given probability distribution. It has been shown that sampling of single haplotypes from each of many demes should yield genealogies with similar properties as belonging to random mating populations (Wakeley, 1999, 2001; Wakeley and Aliacar, 2001).Three major haplogroups were recognized: blue (B), green (G) and red (R) (see below). Genetically non-admixed regions representing each haplogroup were selected to estimate the divergence times between the genetic clusters. The B–G-haplogroup separation was estimated by comparing region 3 with regions 7 and 8 (Fig. 2), the B–R separation by comparing regions 3 and 10, and the R–G-haplogroup separation by comparing regions 7 and 8 with region 10. The R–G analysis should be treated cautiously, as the B haplogroup is more similar to both R and G, than R and G are to each other, violating the IM assumption that no populations should be more genetically similar to the populations implemented in the model than to each another.

Three separate runs were performed for each pairwise comparison, one short (run time ≥1 h) and two long (run time long enough to achieve at least 30 million steps in chains after a burn-in of 106 steps), with minimum effective sample size (ESS) recorded being 138, and most ESS values being >1000. The average B–G estimate is based on four IM runs, the B–R estimate is based on two IM runs, while the R–G estimate is based on four IM runs with similar prior values but different random number seeds. Average substitution rates was set to 1·3 × 10−9 per nucleotide (Richardson et al., 2001), average generation time was arbitrarily set to 5 years (which does not influence divergence time estimates), and historical migration rates was set to zero to reduce the number of parameters in the model. Since no migration is assumed, the estimated divergence times between haplogroups should be viewed as minimum estimates. In the initial, short IM runs, upper prior values of the mutation and divergence time parameters were set to 100. In the two following long runs, prior values were adjusted according to initial results, ensuring that the complete likelihood profiles were included. Prior values for the various analyses are shown in Supplementary Data Table S3.

RESULTS

Haplotype diversity and distribution

The total chloroplast trnL-F dataset comprised 483 accessions of A. alpina sampled from 296 locations, and 71 trnL-F types were recognized, 34 of which are newly discovered (types 38–71). The alignment positions 419–438 comprised a complex variable microsatellite C(1–5)T(8–12) (Supplementary Data Table S2), the size of which varied independently among unrelated sequences. After excluding this region, 57 unique haplotypes were recognized (Supplementary Data Table S2). A network consisting of three major haplogroups was obtained for the unique sequences and these are coloured blue (B), green (G) and red (R) in Fig. 2.

The samples were then organized into ten groupings, reflecting mountain systems and observed haplotype distributions (Fig. 2A and Supplementary Data Table S1). Samples from Levant (Mt Lebanon complex) and central Anatolia were pooled (region 6) due to a sharing of rare related green and red haplotypes (Supplementary Data Table S1). The global distribution of haplogroups is given in Fig. 2A, and detailed distributions for Anatolia and eastern Africa are given in Fig. 2C, D. The B haplogroup occurs throughout the western Anatolian–European–Arctic parts of the species range, the G haplogroup occurs in the eastern Anatolian–Caucasus–Iranian Plateau–Arabian Peninsula part, but also enters the northern Ethiopian mountains (Fig. 2D). The R haplogroup mainly occurs in the eastern African part of the species range (red lineage; Fig. 2B, D), but is narrowly distributed in southern parts of central Anatolia and Levant (pink and light orange lineages; Fig. 2C). central Anatolia/Levant is the only part of the species range were all three haplogroups occur, including the three important haplotypes B1, G1 and R1, which link the three haplogroups together (Fig. 2B, C).

Phylogeography and population genetics

The dataset was structured into ten geographic groups (Table 1). Only four haplotypes, G6, R6, R9 and B1, were widespread. Thirty-two of the 57 haplotypes were private to single accessions (Appendix I), with 50 private to single regions. Central Anatolia was the most genetically diverse region, having the greatest haplotype diversity in absolute terms, with 13 of 57 haplotypes, the highest haplotype richness at R = 6·378 and the highest nucleotide diversity at π = 0·423 % (Table 2), exceeding the pooled global population value at π = 0·319 %. Haplotype B1 dominates throughout the B-haplogroup range, and western Anatolia had the highest haplotype and nucleotide diversity within the B haplogroup (Table 2). Both the haplotypic and nucleotide diversity reduced progressively from south-east Europe towards the Arctic (regions 4–1 in Fig. 2A). Haplotype G6 dominates throughout the G-haplogroup range, with regions 7 and 8 (Fig. 2A) having similar low values of haplotype richness and nucleotide diversity (Table 2). Haplotypes R6 and R9 are common in Ethiopia and eastern Africa, the two main areas of the R-haplogroup range. The Ethiopian mountains had substantially higher haplotypic and nucleotide diversity compared with the rest of eastern Africa (Table 2), reflecting the local mixing for G and R haplotypes (region 9; Fig 2D).

Table 1.

Distribution of haplotypes among the ten geographic regions and numbers of individuals (n) used for genetic analysis

Region n B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 B11 B12 B13 B14 B15 B16 B17 B18 B19 B20 B21 B22 B23 B24 B25 B26 B27 B28 B29 B30 G1 G2 G3 G4 G5 G6 G7 G8 G9 G10 G11 G12 G13 G14 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 R11 R12 R13
Arctic 22 21 . . . . . . . . . . . 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Western and central Europe 65 52 . 6 . 1 . . . . . . 2 . . . 1 . . 1 . 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
North Africa and Macronesia 26 20 2 . . . . . . . . . . . . 1 . 1 . . . . . . 1 . . . . . 1 . . . . . . . . . . . . . . . . . . . . . . . . . . .
South-east.Europe 149 118 . . 1 . 1 1 1 2 1 6 . . 2 . . . . . 2 . 1 . 11 . . 1 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Western Anatolia 21 12 . . . . . . . . . . . . . . . . 1 . . . . . . 1 5 . . 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Central Anatolia 44 7 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 5 1 1 3 18 . . . . . . . . 1 1 1 2 1 . . . . . . . .
Eastern Anatolia and Iranian Plataeux 58 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 2 . 1 1 1 1 . . . . . . . . . . . . . . .
Arabian penisula 16 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 . . . . . . 1 1 . . . . . . . . . . . .
Ethiopia 44 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 . 2 . . . . . . . . . . . 7 . 2 16 5 . . 1
EasternAfrica 39 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 3 . 12 . 1 1 1
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Table 2.

Distribution of haplotype diversity by sampling region

Haplogroup frequencies
 R
Region n n blue = n green = n red = Total (n haps) n haps private individual n haps private regional % abundence n haps regionally private 16 74 π
(1) Arctic and North America 22 22 0 0 2 1 1 4·545 0·727 – 0·013
(2) Western-central European 65 65 0 0 7 3 6 20·000 2·438 – 0·060
(3) North Africa and Macronesia 26 26 0 0 6 4 3 11·538 3·323 – 0·077
(4) South-eastern European 149 149 0 0 14 8 12 13·422 2·698 – 0·083
(5) Western Anatolia 21 21 0 0 6 4 5 42·857 4·048 – 0·110
(6) Central Anatolia and Levant 44 8 30 6 13 6 10 40·909 6·378 – 0·423
(7) Caucasus and Iranian Plateau 58 0 58 0 6 4 5 10·344 1·583 – 0·034
(8) Arabian Peninsula 16 0 16 0 3 2 2 12·500 2·000 – 0·036
(9) Ethiopia 44 0 13 31 7 3 3 20·000 4·440 – 0·430
(10) Eastern Africa 39 0 0 39 6 2 3 13·158 3·037 – 0·113
Blue regions 1–5 283 283 0 0 31 20 28 20·922 – 11·314 0·077
Green regions 7–8 74 0 74 0 8 6 8 9·459 – 7·000 0·035
Red regions 9–10 83 0 13 70 10 2 9 86·747 – 8·750 0·313
Red regions 9–10 (no green haplotypes) 70 0 0 70 8 1 8 100·000 – – 0·130
World-wide (regions 1–10) 484 291 117 76 57 32 – – – – 0·3187
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Sample size (n), frequencies of haplotypes by haplotype clades, numbers of haplotypes, private haplotypes, haplotype diversity by rarefraction (R) for corrected for smallest sample size at regional (n = 16) and haplogroup levels (n = 74), nucleotide diversity (π).

Regions 1–10 are shown in Fig. 2.

At a global scale a significant proportion of genetic variation was partitioned among populations, and FST was 0. 438 (P = 0·0001) when based solely on haplotype frequencies, and ΦST was 0·631 (P = 0·0001) when pairwise haplotype genetic distances were also considered. GST and NST are analogous measures to FST and ΦST and were estimated to be 0·439 and 0·557, respectively, and NST was significantly larger (P = 0·05), indicating related haplotypes are geographically arranged. Again, NST (0·521) was significantly higher than GST (0·357) for the central Anatolian diversity hotspot and the neighbouring regions (regions 5–7), indicating local haplotype diversity is geographically arranged in this critical area (P = 0·01).

Genetic differentiation among regions outside of the central Anatolia diversity hotspot was considerable ΦST = 0·773 (P = 0·0001), and this was mainly due to the differences among the main B/G/R-haplogroup ranges ΦST = 0·745 (P = 0·0002), rather than due to differences within B/G/R-haplogroup ranges. This was supported by individual AMOVA tests on the combined regions of the B-haplogroup range (ΦST = 0·041, P = 0·0001), the G-haplogroup range (ΦST = 0·006, P = 0·3450) and the R-haplogroup range (ΦST = 0·183, P = 0·0001). There is little genetic differentiation among populations from south-east Europe and western Anatolia (regions 4 and 5, ΦST = 0·095, P = 0·005), which are separated by the Sea of Marmara (Fig. 2A, C). Conversely, populations from central Anatolia and the eastern Anatolia–Iranian Plateaux had higher genetic differentiation (regions 6 and 7; ΦST = 0·333, P = 0·000), despite greater geographic connectivity via the Anatolian diagonal mountain systems.

Demographic history and divergence

Separate mismatch analyses on the B and G haplogroups, including the representative Anatolia diversity, did not reject the sudden expansion model (P = 0·710, P = 0·660), and both followed a uni-modal mismatch distribution (Fig. 3A, B). The sudden expansion model was rejected for R-haplogroup diversity (P = 0·050; Fig. 3C), and was also rejected when the African R-haplotype diversity was analysed separately (P = 0·001; Fig. 3D). Mismatch analysis on the data derived from within Anatolia did not reject the sudden expansion model for either the G haplogroup (P = 1·000), or the B haplogroup (P = 0·270). Insufficient data (n = 6) prevented a meaningful test for the Anatolia R-haplogroup diversity.

Fig. 3.

Fig. 3.

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Mismatch distribution analyses of pairwise haplotype differences, observed values, expected values (as indicated), for European (blue) lineages (A), Near East (green) lineages (B), Asian–African (red) lineages (C) and African only (red) lineages (D).

The IM analyses resulted in convergence of parameter estimates, with high ESS values, almost identical distributions of all pairs of long-runs, and posterior estimates not near the edge of uniform prior values. The divergence time probability distributions were estimated for all pairwise combinations of haplogroups (Fig. 4): the B–G haplogroups had the highest probability for t = 0·66 Mya (averaged over four runs, 95 % CI between 0·25 and 3·86 Mya), the B–R haplogroups had the highest probability for t = 0·73 Mya (averaged over two runs 95 % CI between 0·23 and 2·48 Mya), and the highest probability for the R–G-haplogroup separation was t = 2·69 Mya (averaged over four runs 95 % CI between 0·80 and 6·43 Mya). The latter included the haplo-groups with the greatest nucleotide distances, and therefore represents an estimate for the overall age of extant haplotype diversification.

Fig. 4.

Fig. 4.

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Results of the IM runs, comparing divergence time estimates between haplogroup pairings as indicated, based on the sampling regions outlined in Fig. 2A, and haplotype information given in Table 1. Time since divergence is measured in millions of years since present.

DISCUSSION

This study confirms the importance of Anatolia and associated Levant in understanding the global diversification history of the arctic–alpine Arabis alpina. From the dense sampling in the present study, extensive genetic diversity was recovered, including substantial new chloroplast sequence variation not recovered by previous studies (Koch et al., 2006; Assefa et al., 2007; Ansell et al., 2008), despite these studies representing much of the world-wide range. In total the number of haplotypes increased from 31 to 57 (Supplementary Data Table S2) and, remarkably, on average one new haplotype was discovered for every 8·5 samples at the global scale, increasing to every 3·6 samples in Anatolia. All three previously described haplogroups and their global distributions were recovered (Koch et al., 2006; Assefa et al., 2007), and their distributions were confirmed to extend into the interior of Anatolia. Furthermore, it was shown that the genotypic and nucleotide diversity of Anatolia, especially central Anatolia (including Levant), exceeded that of all other areas.

Centres of genetic diversity have often been associated with range expansion and population amalgamation (Petit et al., 2003; Walter and Epperson, 2005). The present results, however, strongly indicate that Anatolia is the cradle of diversity. First, there was no narrowly delimitated range of haplogroup frequency shift, as typified from hybrid zones of trees (Ferris et al., 1998; Bartish et al., 2006), or by grasshoppers, shrews and various bats from central Anatolia, which have divergent European and Near East lineages (Cooper et al., 1995; Bilgin et al., 2006, 2008; Furman et al., 2009). Secondly, the Anatolia haplotype diversity includes the most common European and Near East haplotypes (B1, G6), plus all intermediate haplotypes linking the European, Near East and eastern African lineages (Fig. 2B, C). Of the remaining genealogically important haplotypes, only the red eastern African diversity is missing from Anatolia, and these probably originated in situ due to geographic isolation from the main species range (Koch et al., 2006; Assefa et al., 2007). Hence the prsent results indicate thatAnatolia is the centre of origin for A. alpina, with a long history of lineage accumulation, functioning as a major Pleistocene refugium.

Species origin

The evidence presented is consistent with previous hypotheses of (1) Anatolia being the cradle for Brassicaceae diversification (Koch and Kiefer, 2006; Franzke et al., 2009) and more specifically, (2) that the genus Arabis originated in the Mediterranean or Turko-Iranian areas (Meusel et al., 1965; Hedge, 1976). A molecular phylogeny of the genus Arabis (R. Karl et al., University of Heidelberg, Germany, unpubl. res.) supports these conclusions and confirms the B haplogroup is ancestral for A. alpina. Within the B-haplogroup range western-Anatolia had the highest genetic diversity (Table 2), suggesting that Arabis alpina may have originated in this region. An alternative ‘out of Africa’ scenario is not supported, as the ancestral R-haplotype (R1) and descendents (R2–R5) are private to the areas covering central Anatolia and Levant.

For alpine species, the availability of cold habitats are influential on species and population ranges and the accumulation of genetic diversity (Comes and Kadereit, 2003). Using coalescence analysis, it was estimated that divergence between the most distantly related haplogroups (R, G) commenced around 2·69 Mya (95 % CI 0·80–6·43 Mya). These results must be interpreted cautiously, reflecting the single gene approach of the present study, and the violation of IM assumption, specifically that no other populations are more closely related to the sampled populations than they are to each other (R–G vs. R-B or G-B comparisons). Interestingly, our estimate is similar to a previous estimate of 2·1 Mya estimated from synonymous mutation rates (Koch et al., 2006), and approximates to an origin at the Pliocene–Pleistocene transition, when the climate rapidly cooled (Webb and Bartlein, 1992) and suitable alpine growing conditions would have become more widespread. The subsequent haplogroup diversifications were estimated around 0·70 Mya and roughly coincided with the onset of the Cromerian Complex of glaciation cycles (approx. 300–850 Kya), when the Anatolian climate was 4–5 °C cooler than at present (Erinç, 1978; Furman et al., 2009). Similar divergence times were derived for the separation of Euro-Anatolian/Near Eastern lineages of yellow-necked house mouse, the white-toothed shrew and oak gallwasps (Michaux et al., 2004; Dubey et al., 2006; Challis et al., 2007), suggesting the widespread involvement of early Middle Pleistocene climate fluctuation on the diversification of Anatolian biota.

Pleistocene glacial survival and the accumulation of diversity

Anatolia's dissected mountainous landscape has been crucial for the long-term survival of A. alpina and its ability to accumulate genetic diversity. There was no major Pleistocene ice-shield equivalent to those covering the European Alps or Scandinavia and only the higher mountain peaks above 2200 m were glaciated (Erinç, 1978; Atalay, 1996), providing opportunities for local population survival. Furthermore, the mountain systems provided moist conditions at higher elevations, offsetting the drier climate that prevailed during the glacial periods (Webb and Bartlein, 1992). Alpine species like A. alpina would have potentially had broad glacial distributions in Anatolia, albeit around the alpine belt as a fragmented network of local survival centres. This is especially likely in the more moist coastal Taurus Mountains of southern Anatolia (Davis, 1971; Ekim and Güner, 1986). The situation would have permitted localized altitudinal migration responses to the Pleistocene temperature fluctuations within a shifting alpine belt, thereby mostly retaining local diversity structures. This is analogous to the ‘phalanx’ migration model (Ibrahim et al., 1996) more often applied to the survival of temperate biota in the southern European mountain refugia (Hewitt, 2004; Schmitt, 2007).

For European alpines there are extensive palaeo-ecological, geological and fossil data to support the developed phylogeographic scenarios (Tribsch and Schönswetter, 2003; Comes and Kadereit, 2003). Similar data are currently lacking for Anatolian alpines, and we must rely on genetic data and Pleistocene glacial dynamics to support our hypothesis. It is reasonable to assume that the availability of suitable habitats becomes limited for alpines during the warm inter-glacial phases in Anatolia. Regular range fragmentation during the inter-glacials (geographic isolation) may potentially restrict genetic exchange and promote the accumulation of local genotypes among the surviving populations. This should result in the widespread distribution of local genotypes and for related genotypes to be spatially clustered. Haplotypes private to single localities were scattered throughout the Anatolian range of A. alpina (Fig. 2C), and genealogically related haplotypes (G2–5, R2–4) were locally arranged in the southern Anti-Taurus and Mount Lebanon complexes (Fig. 2C). Notably, haplotype B26 was distributed among the southern Taurus Mountain localities (Fig. 2C), which are also important for their endemic Arabis species (R. Karl et al., University of Heidelberg, Germany, unpubl. res.) and locally differentiated lineages of Lebanese cedar and ground squirrels (Gündüz et al., 2007; Fady et al., 2008). Furthermore, the southern Anatolian systems were recently recognized as molecular diversity hotspots (Médail et al., 2009), and local ‘micro-refugia’ for pond turtles (Fritz et al., 2009), again consistent with a hypothesis of multiple survival centres.

Out of Anatolia

Most strikingly the internal haplogroup arrangement of Anatolia closely resembles the wider global pattern for A. alpina (Koch et al., 2006) (Fig. 2C). Hence, we argue that the global haplogroup arrangement was achieved by expansions from already internally structured Anatolian diversity. This ‘inside-out’ scenario is compatible with an existing ‘three-times out of Asia Minor’ hypothesis for A. alpina (Koch et al., 2006), but we note that the G1-dominated Arabian Peninsula and Caucasus/Iranian Plateau are separated by G2- to G5-dominated intervening Anti-Taurus/Mt Lebanon ranges (Fig. 2C). This suggests these regions were independently colonized, and thus a ‘four-times out of Anatolia’ scenario seems likely.

Two geographic features are widely acknowledged to have influenced the expansions of plants and animals around Anatolia: the Sea of Marmara and associated Thyracian Plain in European Turkey, and the high ‘Anatolian diagonal’ mountains in the east (Davis, 1971; Ekim and Güner, 1986; Rokas, et al., 2003; Bilgin et al., 2009). The hills around the Sea of Marmara reach only 1100 m, and currently have a Mediterranean climate and flora (Davis, 1965–85), but dry steppe communities dominated this area during Pleistocene glaciations (Michaux et al., 2004). Pleistocene land bridges regularly formed across the Sea of Marmara (Kerey et al., 2004; Magyari et al., 2008) and, for some temperate animal species there is clear evidence of inter-continental gene flow (Dubey et al., 2007; Furman et al., 2009; Stamatis et al., 2009). In contrast, for alpine plant species, the current and past ecological conditions around the Sea of Marmara potentially represent a substantial geographic/ecological barrier for inter-continental gene flow. Surprisingly, related B-haplotypes were found distributed in both western Anatolia and the southern Balkans and there is negligible genetic differentiation between these two areas (ΦST = 0·095). Hence we are forced to conclude that the Sea of Marmara region has not been a historical ecological barrier for A. alpina. Inter-continental genetic exchange most likely occurred during favourable glacial conditions, when the alpine habitats were presumably at lower elevations, creating greater ecological connectivity between the two continents. Furthermore, the progressive loss of nucleotide diversity from western Anatolia to northern Europe (Table 2) suggests that gene flow has predominantly occurred from Anatolia into Europe.

The mountains of the ‘Anatolian diagonal’ in eastern Anatolia are substantially different (>2000 m), providing extensive areas for species preferring cool moist climates to grow at higher elevations. Indeed, the diagonal is characterized by numerous plant and insect endemics (Davis, 1971; Ekim and Güner, 1986; Çiplak, 2003), which may have persisted locally or nearby during recent glaciations (Gündüz et al., 2007). Importantly the diagonal mountains have a north-east/south-west orientation, whereas most Anatolian mountain systems have east–west orientations, and this provides a continuous mountain chain connection between the Mediterranean and Caspian seas. This links the southern Taurus diversity hotspot (Médail and Diadema, 2009) to nearby Caucasus and Near East mountain systems. Thus, we postulate the diagonal has been a migratory corridor for A. alpina range expansion out of Anatolia during favourable glacial periods. This is supported by populations near to the Mediterranean Sea harbouring haplotypes sister to those commonly detected from populations in the Caucasus and Iranian Plateau. Mismatch tests further indicated a history of sudden range expansion for populations encompassing the diagonal and the adjacent eastern Anatolia. We note that for several temperate animal species the diagonal does function as a barrier to lineage mixing (Rokas, et al., 2003; Dubey et al., 2006; Bilgin et al., 2009), and the present contrasting results for an alpine species highlight the importance of individual ecologies when interpreting mountains as barriers or corridors.

Conclusions

Despite the dominating influence of mountain systems on the biogeographic structure of Anatolia, the phylogeographic histories of mountain species are poorly known in this biodiversity hotspot. The prsent study demonstrates the importance of the Anatolia mountains as the centre for global diversification for arctic–alpine A. alpina, that the local Anatolian diversity pattern mirrors the global pattern, and that local Pleistocene population history has left a genetic imprint on the global population structure. Relatively dense and even sampling was crucial for recovering critical genotypes in southern Anatolia to substantiate these findings. Currently there are few detailed studies of intra-specific genetic diversity from other plants to reliably assess Anatolia's Pleistocene history as a centre of gene pool amalgamation or centre of diversity (Kučera et al., 2006; Gömöry et al., 2007; Naydenov et al., 2007). We were fortunate to have access to the large herbarium legacy of the Flora Turkey project (Davis, 1965–85) as a molecular resource to discriminate between these alternative interpretations for A. alpina. Consequently our study also illustrates the importance of sampling and scaling effects when conducting phylogeographic analysis, especially in complex regions of lineage and flora mixing.

SUPPLEMENTARY DATA

Supplementary data are available online at www.aob.oxfordjournals.org and consist of the following. Table S1: Sampling information. Table S2: Haplotype alignments and classifications.Table S3: Prior values used for the various IM program analyses.

Supplementary Data
supp_108_2_241__index.html (1.2KB, html)

ACKNOWLEDGEMENTS

We thank Mary Gibby and Andreas Hemp for collecting material in Yemen and eastern Africa, Cathy Smith for geo-referencing, Bob Press for comments on the manuscript development, Maria Albani and George Coupland for support during fieldwork in Greece, and Julia Llewellyn-Hughes and her team for the NHM DNA sequencing service. We are grateful to the curators of the herbaria B, BM, CO and E for allowing us to use leaf samples from herbaria sheets in our study. The project was financially supported by the Department Botany at the NHM London, and by the Swedish and Norwegian Research Councils to H.K.S.

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supp_108_2_241__index.html (1.2KB, html)
supp_mcr134_mcr134supp_table1.xls (152KB, xls)
supp_mcr134_mcr134supp_table2.xls (97.5KB, xls)
supp_mcr134_mcr134supp_table3.doc (30KB, doc)

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