Structure and Evolution of the Mitochondrial DNA Complete Control Region in the Lizard Lacerta dugesii (Lacertidae, Sauria)

We sequenced the complete control region (CR) and adjacent tRNAs, partial 12S rRNA, and cytochrome b (over 3100 bp) from eight individuals of Madeiran wall lizards, Lacerta dugesii, from four distinct island populations. The tRNAs exhibit a high degree of intraspecific polymorphisms compared to other vertebrates. All CR sequences include a minisatellite that varies in length between populations but is apparently fixed within them. Variation in minisatellite length appears between populations separated by apparently very short evolutionary time spans. Many motifs identified in the CR of other vertebrates are not highly conserved, although conserved blocks are identifiable between the few published reptile CR sequences. Overall there are extensive differences in the internal organization of the reptile CR compared to the more widely studied mammals and birds. Variability in the CR is lower than in cytochrome b, but higher than in 12S rRNA. Phylogenetic analysis of these sequences produces a well-resolved estimate of relationships between populations.


COMPLEMENTARY MATERIAL
The following material was included in early versions of the manuscript but was cut off in later revisions. Nevertheless, it is intended to help those wishing to better understand the structure and evolution of Lacerta dugesii's mitochondrial DNA.
PCR cycle conditions were 30 sec at 94ºC, 30 sec at 60ºC and 2 min at 70ºC, for 35 cycles.
Following the sequencing of this fragment, nine primers were successively designed from conserved regions among the 8 sequences, to amplify consecutive segments, until sequences from both extremities overlapped (primer-walking strategy). Primer sequences and their relative locations are as follows: Amplified fragments were always sequenced three times and in both directions, on a 377 Applied Biosystem DNA Sequencing Apparatus, with the same set of primers used for amplification. One 10-mer primer (cBLint5-5'TTGCCTCCAC3') was specifically designed to work with primer 12Lint5. These two primers were used to amplify the region containing a repeat motif. We amplified this fragment in ten individuals from each site, to check for variation of number of repeats within populations.

The tRNAs
Here we present additional material regarding the tRNA sequences found in L.
dugesii as well as in comparisons with other animals. Particularly in relation to tRNA Pro it is true that not all forms present the four stems usually present in tRNAs, some having shifted the TΨC stem for a bigger replacement loop. Most of these tRNA forms present one or two

The Control Region (CR)
Lacerta dugesii CR is depicted schematically in Figure 4 of Brehm et al. (2003), but here we include the CR -Complete Sequence (D1 of one individual analysed (D1, Deserta Isl.) so it should make much more easier to follow the scheme:

The Minisatellite
As

CBS (Conserved Block Sequences)
Bellow we show the best alignment of sequences including the Conserved Sequence Blocks 1-3 (CSB1-3) in a lizard (L. dugesii, this work), a skink (Eumeces egregious, Kumazawa and Nishida 1999), a iguanidae (Iguana iguana, Janke et al. 2001), a turtle (Kinosternon hirtipes GenBank AF316136), a snake (Dinodon semicarinatus, Kumazawa et al. 1998), a gull and a chicken (Crochet and Desmarais 2000), two partridges (Alectoris barbara and A.graeca, Lucchini 1998) andsix mammals (Rattus andMus, Brown et al. 1986; Glis glis, Sbisà et al. 1997;Canis familiaris, Rothuizen, et al. direct submission to GenBank X97343; Canis lupus, Vila et al.1997;Equus caballus, Ishida et al. 1994). In the alignment we did not always use the CSB boundaries proposed by Brown et al. (1986) and instead used the complete sequences that gave a better score on bp matching when compared with reptile sequences. An asterisk denotes that among several options for CSB-1 element, that was the best match. Bases common to all vertebrates are underlined. We present here an alternative sequence for Alectoris graeca from the one proposed by Randi and Lucchini (1998). We could not find any such pattern for Ursus arctus.