Additional file 1: of Mind the gap! The mitochondrial control region and its power as a phylogenetic marker in echinoids

Figure S1. Gel image showing PCR products. Figure S2. Sequencing results shown as a four-color chromatogram. Figure S3. Substitution saturation plots. Table S1. Substitution saturation analysis. (PDF 1735 kb

MOESM1 of DNA analysis of molluscs from a museum wet collection: a comparison of different extraction methods

Additional file 1. In the additional file a detailed list of all samples used in this study can be found, including sample age and the inventory numbers. Furthermore, the DNA concentration [ng/Âľl] for both extraction methods (Promega-THK and the Gen-ial-ATK) and the formaldehyde content [mg/l] are given. The PCR success of all samples is indicated as well

In Search of Glacial Refuges of the Land Snail <i>Orcula dolium</i> (Pulmonata, Orculidae) - An Integrative Approach Using DNA Sequence and Fossil Data

<div><p>Harboring a large number of endemic species, the Alps and the Western Carpathians are considered as major centers of biodiversity. Nonetheless, the general opinion until the turn of the millennium was that both Central European mountain regions did not provide suitable habitat during the Last Glacial Maximum, but were colonized later from southern refuges. However, recent molecular genetic studies provide new evidence for peripheral Alpine refuges. We studied the phylogeography of the calciphilous land snail <i>O. dolium</i> across its distribution in the Alps and the Western Carpathians to assess the amount of intraspecific differentiation and to detect potential glacial refuges. A partial sequence of the mitochondrial COI was analyzed in 373 specimens from 135 sampling sites, and for a subset of individuals, partial sequences of the mitochondrial 16S and the nuclear histone H3 and H4 were sequenced. A molecular clock analysis was combined with a reconstruction of the species’ geographic range history to estimate how its lineages spread in the course of time. In order to obtain further information on the species’ past distribution, we also screened its extensive Pleistocene fossil record. The reconstruction of geographic range history suggests that <i>O. dolium</i> is of Western Carpathian origin and diversified already around the Miocene-Pliocene boundary. The fossil record supports the species’ presence at more than 40 sites during the last glacial and earlier cold periods, most of them in the Western Carpathians and the Pannonian Basin. The populations of <i>O. dolium</i> display a high genetic diversity with maximum intraspecific <i>p</i>-distances of 18.4% (COI) and 14.4% (16S). The existence of various diverged clades suggests the survival in several geographically separated refuges. Moreover, the sequence patterns provide evidence of multiple migrations between the Alps and the Western Carpathians. The results indicate that the Southern Calcareous Alps were probably colonized only during the Holocene.</p></div

Distribution of localities investigated in the Alps and the Western Carpathians.

<p>The symbols indicate the presence of the respective mt clades. If specimens of different mt clades are present at the same localities, the respective clade symbols are shown encircled with a line pointing towards the locality. A picture of the shell of a type specimen of <i>O. dolium</i> (Syntype NHMW 14765/1820.26.61/2; size: 6.8 mm in height) is provided in the left upper corner.</p

Mean and maximum genetic <i>p</i>-distances (in %) for the 16S sequences.

<p>Mean and maximum genetic <i>p</i>-distances (in %) for the 16S sequences.</p

Median Joining network of the H4/H3 sequences.

<p>Bars indicate substitutions within the H3, H4 and the spacer region. The loss or gain of bases (indels) in the spacer region is displayed by arrows pointing in the respective direction and the numbers indicate in how many bases the haplotypes/alleles differ from each other. The three most common alleles are named as HT1, HT2 and HT3. The size of the circles corresponds to the number of sequences providing the same allele. The colors correspond to one of the mountain regions defined in Fig. 1. To facilitate the comparison of nc and mt data, the specimen labels and the clade affiliation are indicated next to the haplotype circles.</p

Distribution of fossil <i>O. dolium</i> in Central Europe during warm (A) and cold (B) Pleistocene climate stages.

<p>The LGM glacier line indicates the maximum extent of glaciers during the Weichselian (35 to 19 ka). The grey-shaded areas correspond to the current distribution of <i>O. dolium</i>.</p

BI tree of the concatenated COI and 16S sequences.

<p>Posterior probabilities and ML bootstrap values are provided for all nodes above sub-clade level. The scale bar indicates the expected number of substitutions per site according to the model of sequence evolution applied. The dots indicate nodes with posterior probabilities of 1.0 and bootstrap values of more than 98% (black) or 95% to 97% (grey) in the ML analyses, respectively.</p

Primer sets for amplification and sequencing.

<p>The annealing temperatures are provided for the Finnzymes Phusion and the RocheTaq Polymerase, respectively.</p

BI analysis of COI haplotypes.

<p>The central tree provides an overview. Posterior probabilities are given at the main nodes. Black dots indicate nodes with maximum support. A specimen of <i>O. conica</i> is used as outgroup. More details are provided in the partial trees with different colors corresponding to the geographic mountain regions depicted in Fig. 1. The scale bars indicate the expected number of substitutions per site according to the model of sequence evolution applied.</p