Ancient DNA from historical and subfossil wood has a great potential to provide new insights into the history of tree populations. However, its extraction and analysis have not become routine, mainly because contamination of the wood with modern plant material can complicate the verification of genetic information. Here, we used sapwood tissue from 22 subfossil pines that were growing c. 13 000 yr bp in Zurich, Switzerland. We developed and evaluated protocols to eliminate surface contamination, and we tested ancient DNA authenticity based on plastid DNA metabarcoding and the assessment of post-mortem DNA damage. A novel approach using laser irradiation coupled with bleaching and surface removal was most efficient in eliminating contaminating DNA. DNA metabarcoding confirmed which ancient DNA samples repeatedly amplified pine DNA and were free of exogenous plant taxa. Pine DNA sequences of these samples showed a high degree of cytosine to thymine mismatches, typical of post-mortem damage. Stringent decontamination of wood surfaces combined with DNA metabarcoding and assessment of post-mortem DNA damage allowed us to authenticate ancient DNA retrieved from the oldest Late Glacial pine forest. These techniques can be applied to any subfossil wood and are likely to improve the accessibility of relict wood for genome-scale ancient DNA studies.

Genetic differentiation of scattered populations at neutral loci is characterized by genetic drift counteracted by the remaining gene flow. Populations of Pinus cembra in the Carpathian Mountains are isolated and restricted to island-like stands at high-elevation mountain ranges. In contrast, paleobotanical data suggest an extended early Holocene distribution of P. cembra in the Carpathians and its surrounding areas, which has contracted to the currently disjunct occurrences. We analyzed the genetic variation of 11 Carpathian populations of P. cembra at chloroplast and, in part newly developed, nuclear microsatellites. Both marker types revealed low levels of genetic differentiation and a lack of isolation by distance, reflecting the post-glacial retraction of the species to its current distribution. Stronger effects of genetic drift were implied by the higher genetic differentiation found for haploid chloroplast than for diploid nuclear markers. Moreover, we found no association between the values of population genetic differentiation for the two marker types. Several populations indicated recent genetic bottlenecks and inbreeding as a consequence of decline in population sizes. Moreover, we found individuals in two populations from the Rodnei Mountains that strikingly differed in assignment probabilities from the remaining specimens, suggesting that they had been introduced from a provenance outside the studied populations. Comparison with Eastern Alpine P. cembra and individuals of the closely related Pinus sibirica suggests that these individuals presumably are P. sibirica. Our study highlights the importance of the maintenance of sufficiently large local population sizes for conservation due to low connectivity between local occurrences.

Tertiary relict plant species of Europe have had a large distribution range before the Pleistocene but today are confined to small refugial areas. Syringa josikaea of the largely East Asian genus Syringa is a shrub of temperate forests in the Carpathians, restricted to altogether 25 small populations in two disjunct areas, the Apuseni Mountains (Romania) and the Ukrainian Carpathians. Miocene and Pleistocene fossil remains indicate the long‐term presence of the species in Central Europe; hence S. josikaea has been considered a Tertiary relict. We aimed at clarifying the historical biogeography of S. josikaea by estimating the divergence time between S. josikaea and its Asian relatives, and by analysing intraspecific variation using multiple DNA sequences as well as microsatellites. The estimated divergence time between S. josikaea and its closest relatives based on nuclear ribosomal DNA sequences is 1.88 Mya [0.30–4.04 Mya highest posterior density], suggesting a relatively recent disruption of a formerly continuous distribution area. This time corresponds to the period of Early Pleistocene extinctions, when many Tertiary plant taxa went extinct in Europe. Sequence variation was found to be very low within S. josikaea. Complete identity among all samples in cpDNA may imply a bottleneck. Four different ribotypes found showed no geographic differentiation between the Apuseni Mountains and the Ukrainian Carpathians, and differentiation between these two areas was weak when considering microsatellite variation. Together these observations may imply that the extant disjunct distribution of the species arose only recently, either through colonization from one glacial refugial area or from disruption of one such area. © 2015 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, ●●, ●●–●●.

The species of the genus Syringa L. are among the most popular ornamental plants worldwide. One particular species, Syringa josikaea, a rare endemic of the Carpathian Mountains, is of great conservation interest. Although microsatellite markers may be useful for studying the genetic variability of varieties and populations, no microsatellites have previously been characterized for any species of the Syringa genus. Our aim was therefore to test the applicability of microsatellite primers developed for neighboring genera (Olea and Ligustrum) and to complement these with markers isolated and characterized for S. josikaea. Twelve primer pairs of Olea and Ligustrum were tested by optimizing PCR conditions and checking the variability in 40 samples of two populations of S. josikaea. Two of them proved to be easy to PCR amplify and variable at the same time. To develop new primers we constructed a microsatellite enriched library and sequenced 48 clones. 18 sequences contained microsatellite motifs, and three of the designed primer pairs presented high allele variability. The five primer pairs characterized for S. josikaea proved to be highly informative and sufficient to distinguish between individuals. These microsatellite primers are valuable tools to study genetic variation of native populations, genetic lineages of hybrids and cultivars of S. josikaea.

Precious coral species have been used to produce jewelry and ornaments since antiquity. Due to the high value and demand for corals, some coral beds have been heavily fished over past centuries. Fishing and international trade regulations were put in place to regulate fishing practices in recent decades. To this date, the control of precious coral exploitation and enforcement of trade rules have been somewhat impaired by the fact that different species of worked coral samples can be extremely difficult to distinguish, even for trained experts. Here, we developed methods to use DNA recovered from precious coral samples worked for jewelry to identify their species. We evaluated purity and quantity of DNA extracted using five different techniques. Then, a minimally invasive sampling protocol was tested, which allowed genetic analysis without compromising the value of the worked coral objects.The best performing DNA extraction technique applies decalcification of the skeletal material with EDTA in the presence of laurylsarcosyl and proteinase, and purification of the DNA with a commercial silica membrane. This method yielded pure DNA in all cases using 100 mg coral material and in over half of the cases when using "quasi non-destructive" sampling with sampled material amounts as low as 2.3 mg. Sequence data of the recovered DNA gave an indication that the range of precious coral species present in the trade is broader than previously anticipated. Precious corals are among the most appreciated and oldest known gems. They are valued for their color, texture and workability (polishing, carving), and have thus been collected and used for adornment for millennia 1-3. Growing demand, particularly in Asia in recent years, has led to an increase in prices of precious corals used in jewelry 4-6. The most valuable precious coral species belong to the Coralliidae family within the Octocorallia subclass of the Anthozoa. The precious coral material used for jewelry is the worked (i.e. cut, carved and polished) hard coral skeletal axis, which is a biogenic material created by a biomineralization process 7. In this process, closely packed magnesium-rich calcite crystals are secreted by coral polyps (1-2 mm in size) to build up a skeleton over decades. The polyps can thrive on the surface of the skeleton as colonies connected and surrounded by a 0.5-1 mm thick surface tissue (coenenchyme) 8. The Coral Commission of The World Jewellery Confederation (CIBJO) lists eight Coralliidae species as significant in the precious coral jewelry industry 9,10. Precious coral products are sold worldwide, with production centers located in Italy, Japan and Taiwan and large-scale trade of raw material between these areas 5,6,11. Until recent decades, the populations of these highly coveted marine animals experienced exploitation in boom and bust cycles where the discovery of precious coral beds led to rushes by coral fishers and these beds were exploited as long as it remained economically feasible 12,13. Local and international regulations were put...

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Ancient plant species surviving in isolated small populations are particularly vulnerable to extinction, therefore understanding their population dynamics is necessary for conservation. The iteroparous perennial relic endemic Ferula sadleriana Ledeb. (Apiaceae) is restricted to seven distant localities in the Carpathian Basin, where it inhabits rocky hills. We monitored the species' largest population on the Pilis Hill, Hungary, over 14-19 years (depending on trait) between 1979 and 2010, and relationships were sought between climatic properties and population attributes. The population of 4000 Ϯ 1509 emergent individuals underwent large interannual fluctuations, with the vegetative stage displaying sevenfold and the reproductive stage twenty-eight-fold differences. Spring and early summer precipitation had a marked influence on abundances and seed set. Alternating years of high and low counts of reproductive plants suggest costs of reproduction that most probably incur prolonged dormancy and retrogression to the vegetative stage. Seed set was positively influenced by number of reproductive plants over years and by plant size within a year. Ungulates nullify yearly reproductive output by grazing on reproductive individuals. This is particularly intense in dry summers, when reproductive output is already low. The strong precipitation response of abundance, absence of clonal propagation and soil seed bank, and geographical isolation of the populations place F. sadleriana at considerable risk under an increasingly variable and extreme climate. Management should seek to maintain the species' original habitat mosaic (potentially compensating for climate variation), minimize grazing damage and anthropogenic disturbance, and establish ex situ conservation programs to provide propagules for eventual reintroduction.