Mitochondrial DNA Variation, but Not Nuclear DNA, Sharply Divides Morphologically Identical Chameleons along an Ancient Geographic Barrier

The Levant is an important migration bridge, harboring border-zones between Afrotropical and palearctic species. Accordingly, Chameleo chameleon, a common species throughout the Mediterranean basin, is morphologically divided in the southern Levant (Israel) into two subspecies, Chamaeleo chamaeleon recticrista (CCR) and C. c. musae (CCM). CCR mostly inhabits the Mediterranean climate (northern Israel), while CCM inhabits the sands of the north-western Negev Desert (southern Israel). AFLP analysis of 94 geographically well dispersed specimens indicated moderate genetic differentiation (PhiPT = 0.097), consistent with the classical division into the two subspecies, CCR and CCM. In contrast, sequence analysis of a 637 bp coding mitochondrial DNA (mtDNA) fragment revealed two distinct phylogenetic clusters which were not consistent with the morphological division: one mtDNA cluster consisted of CCR specimens collected in regions northern of the Jezreel Valley and another mtDNA cluster harboring specimens pertaining to both the CCR and CCM subspecies but collected southern of the Jezreel Valley. AMOVA indicated clear mtDNA differentiation between specimens collected northern and southern to the Jezreel Valley (PhiPT = 0.79), which was further supported by a very low coalescent-based estimate of effective migration rates. Whole chameleon mtDNA sequencing (∼17,400 bp) generated from 11 well dispersed geographic locations revealed 325 mutations sharply differentiating the two mtDNA clusters, suggesting a long allopatric history further supported by BEAST. This separation correlated temporally with the existence of an at least 1 million year old marine barrier at the Jezreel Valley exactly where the mtDNA clusters meet. We discuss possible involvement of gender-dependent life history differences in maintaining such mtDNA genetic differentiation and suggest that it reflects (ancient) local adaptation to mitochondrial-related traits

Neighbor joining trees based on mtDNA sequences from <i>C. chameleon</i> samples.

<p>(A) A tree constructed from 637 bp mtDNA fragments from 57 <i>C. chamaeleon</i> samples in Israel, one each from Portugal and Cyprus, and two from Turkey (Genbank accession numbers are shown). Collection sites are shown for each sample. To assess statistical significance the tree underwent a 1000 bootstrap replicates and scores (percentages) are mentioned near each branch. South –mtDNA genetic cluster including all samples south of the Jezreel Valley (see map in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031372#pone-0031372-g005" target="_blank">Fig 5</a>). North –mtDNA genetic cluster including all samples north of the Jezreel Valley. For similar results using Network analysis – see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031372#pone.0031372.s002" target="_blank">Fig. S2</a>. (B) A tree constructed from 11 whole mtDNA <i>C. chamaeleon</i> samples in Israel which were a subset of the samples analyzed in (A). Bootstrap scores of 1000 replicates are shown near each branch. All phylogenetic analyses were performed using MEGA 4.0.</p

Results of a canonical discriminant function analysis of the morphological data set, demonstrating the separation between the desert (<i>C. c. musae</i>) and Mediterranean (<i>C. c. recticrista</i>) subspecies.

<p>The first canonical variate (score 1 = CV1), which provides the maximal separation among instars, is given by: CV1 = 0.345×logA×0.834×logB−0.013×logD−0.03×logE+0.257×logF+0.121×logG−0.91×logH+0.371×logI−0.45×logJ−1.49×logK+0.354×logL−1.043×logM−0.609×logN+0.83×logO (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031372#pone.0031372.s006" target="_blank">Table S1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031372#pone.0031372.s001" target="_blank">Fig. S1</a>).</p

Analysis of Molecular Variance (AMOVA) of AFLP results^*.

*<p>General PhiTP value calculated from the 2 morphological sub species. PhiPT value is analog to Fst, where values ranging from 0.15–0.25 reflect great genetic differentiation, values ranging from 0.05–0.15 reflect moderate genetic differentiation and under 0.05 suggest little genetic differentiation. AMOVA were perform using Genalex 6.3 for excel 2007. Df – degrees of freedom; SS – sum of square, MS- mean square, Est. Var. – estimated variance.</p><p>Among populations – between the 2 morphological sub species (i.e. between CCR and CCM). Within populations – within <u>each</u> of the 2 morphological sub species (i.e. CCR and CCM).</p

Principal coordinates analysis based on 323 AFLP loci detected in 94 chameleons sampled across different parts of Israel.

<p>Of the total genetic variation in the dataset, 54.3% was explained by the first two coordinates.</p

An integrative map summarizing the result of mDNA, AFLP and morphological analyses conducted for the 94 different chameleons sampled throughout Israel.

<p>An integrative map summarizing the result of mDNA, AFLP and morphological analyses conducted for the 94 different chameleons sampled throughout Israel.</p

Map of chameleon collecting sites across Israel.

<p>1.Avshalom, 2. Nitzana, 3. Kmehin, 4. Shivta Junction, 5. Seher Stream, 6. Ramat Beka, 7. Revivim, 8. Beer Aslug, 9. Nizzanim, 10. Jerusalem, 11. Habonim, 12. Ceasarea, 13. Kishon, 14. Tivon, 15. Salem, 16. Oosha, 17. Gahar mountain, 18. Carmel mountain, 19. Haon, 20. Kochav Hayarden, 21. Fasuta, 22. Ramot, 23. Akbara Stream, 24. Shamir, 25. Korazim, 26. Magen Shaul, 27. Baram, 28. Mazkeret Batia, 29. Avital mountain, 30. Banias, 31. Matat, 32. Rosh HaNikra, 33. Harrit, 34. Bait Shean, 35. Gilboa, 36. Jezreel Junction.</p