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The ability of plants to track seasonal changes is largely dependent on genes assigned to the photoperiod pathway, and variation in those genes is thereby important for adaptation to local day length conditions. Extensive physiological data in several temperate conifer species suggest that populations are adapted to local light conditions, but data on the genes underlying this adaptation are more limited. Here we present nucleotide diversity data from 19 genes putatively involved in photoperiodic response in Norway spruce (Picea abies). Based on similarity to model plants the genes were grouped into three categories according to their presumed position in the photoperiod pathway photoreceptors, circadian clock genes, and downstream targets. An HKA (Hudson, Kreitman and Aquade) test showed a significant excess of diversity at photoreceptor genes, but no departure from neutrality at circadian genes and downstream targets. Departures from neutrality were also tested with Tajima's D and Fay and Wu's H statistics under three demographic scenarios the standard neutral model, a population expansion model, and a more complex population split model. Only one gene, the circadian clock gene PaPRR3 with a highly positive Tajima's D value, deviates significantly from all tested demographic scenarios. As the PaPRR3 gene harbours multiple non-synonymous variants it appears as an excellent candidate gene for control of photoperiod response in Norway spruce. © 2014 Källman et al.

Plant and animal biodiversity can be studied by obtaining DNA directly from the environment. This new approach in combination with the use of generic barcoding primers (metabarcoding) has been suggested as complementary or alternative to traditional biodiversity monitoring in ancient soil sediments. However, the extent to which metabarcoding truly reflects plant composition remains unclear, as does its power to identify species with no pollen or macrofossil evidence. Here, we compared pollen-based and metabarcoding approaches to explore the Holocene plant composition around two lakes in central Scandinavia. At one site, we also compared barcoding results with those obtained in earlier studies with species-specific primers. The pollen analyses revealed a larger number of taxa (46), of which the majority (78%) was not identified by metabarcoding. The metabarcoding identified 14 taxa (MTUs), but allowed identification to a lower taxonomical level. The combined analyses identified 52 taxa. The barcoding primers may favour amplification of certain taxa, as they did not detect taxa previously identified with species-specific primers. Taphonomy and selectiveness of the primers are likely the major factors influencing these results. We conclude that metabarcoding from lake sediments provides a complementary, but not an alternative, tool to pollen analysis for investigating past flora. In the absence of other fossil evidence, metabarcoding gives a local and important signal from the vegetation, but the resulting assemblages show limited capacity to detect all taxa, regardless of their abundance around the lake. We suggest that metabarcoding is followed by pollen analysis and the use of species-specific primers to provide the most comprehensive signal from the environment.

Plant and animal biodiversity can be studied by obtaining DNA directly from the environment. This new approach in combination with the use of generic barcoding primers (metabarcoding) has been suggested as complementary or alternative to traditional biodiversity monitoring in ancient soil sediments. However, the extent to which metabarcoding truly reflects plant composition remains unclear, as does its power to identify species with no pollen or macrofossil evidence. Here, we compared pollen-based and metabarcoding approaches to explore the Holocene plant composition around two lakes in central Scandinavia. At one site, we also compared barcoding results with those obtained in earlier studies with species-specific primers. The pollen analyses revealed a larger number of taxa (46), of which the majority (78%) was not identified by metabarcoding. The metabarcoding identified 14 taxa (MTUs), but allowed identification to a lower taxonomical level. The combined analyses identified 52 taxa. The barcoding primers may favour amplification of certain taxa, as they did not detect taxa previously identified with species-specific primers. Taphonomy and selectiveness of the primers are likely the major factors influencing these results. We conclude that metabarcoding from lake sediments provides a complementary, but not an alternative, tool to pollen analysis for investigating past flora. In the absence of other fossil evidence, metabarcoding gives a local and important signal from the vegetation, but the resulting assemblages show limited capacity to detect all taxa, regardless of their abundance around the lake. We suggest that metabarcoding is followed by pollen analysis and the use of species-specific primers to provide the most comprehensive signal from the environment.

Plant and animal biodiversity can be studied by obtaining DNA directly from the environment. This new approach in combination with the use of generic barcoding primers (metabarcoding) has been suggested as complementary or alternative to traditional biodiversity monitoring in ancient soil sediments. However, the extent to which metabarcoding truly reflects plant composition remains unclear, as does its power to identify species with no pollen or macrofossil evidence. Here, we compared pollen-based and metabarcoding approaches to explore the Holocene plant composition around two lakes in central Scandinavia. At one site, we also compared barcoding results with those obtained in earlier studies with species-specific primers. The pollen analyses revealed a larger numberof taxa (46), of which the majority (78%) was not identified by metabarcoding. The metabarcoding identified 14 taxa (MTUs), but allowed identification to a lower taxonomical level. The combined analyses identified 52 taxa. The barcoding primers may favour amplification of certain taxa, as they did not detect taxa previously identified with species- specific primers. Taphonomy and selectiveness of the primers are likely the major factors influencing these results. We conclude that metabarcoding from lake sediments provides a complementary, but not an alternative, tool to pollen analysis for investigatingpast flora. In the absence of other fossil evidence, metabarcoding gives a local and important signal from the vegetation, but the resulting assemblages show limited capacity to detect all taxa, regardless of their abundance around the lake. We suggest that metabarcoding is followed by pollen analysis and the use of species-specific primers to provide the most comprehensive signal from the environment.

It is commonly believed that trees were absent in Scandinavia during the last glaciation and first recolonized the Scandinavian Peninsula with the retreat of its ice sheet some 9000 years ago. Here, we show the presence of a rare mitochondrial DNA haplotype of spruce that appears unique to Scandinavia and with its highest frequency to the west-an area believed to sustain ice-free refugia during most of the last ice age. We further show the survival of DNA from this haplotype in lake sediments and pollen of Trondelag in central Norway dating back similar to 10,300 years and chloroplast DNA of pine and spruce in lake sediments adjacent to the ice-free Andoya refugium in northwestern Norway as early as similar to 22,000 and 17,700 years ago, respectively. Our findings imply that conifer trees survived in ice-free refugia of Scandinavia during the last glaciation, challenging current views on survival and spread of trees as a response to climate changes.

Ancient DNA has received much attention since the mid-1980s, when the first sequence of an extinct animal species was recovered from a museum specimen. Since then, the majority of ancient DNA studies have focused predominantly on animal species, while studies in plant palaeogenetics have been rather limited, with the notable exception of cultivated species found in archaeological sites. Here, we outline the recent developments in the analysis of plant ancient DNA. We emphasize the trend from species identification to population-level investigation and highlight the potential and the difficulties in this field, related to DNA preservation and to risks of contamination. Further efforts towards the analysis of ancient DNA from the abundant store of fossil plant remains should provide new research opportunities in palaeoecology and phylogeography. In particular, intraspecific variation should be considered not only in cultivated plants but also in wild taxa if palaeogenetics is to become a fully emancipated field of plant research.

Assessments of plant population dynamics in space and time have depended on dated records of fossil pollen synthesized on a subcontinental scale. Genetic analyses of extant populations have revealed spatial relationships that are indicative of past spatial dynamics, but lack an explicit timescale. Synthesis of these data requires genetic analyses from abundant dated fossil material, and this has hitherto been lacking. Fossil pollen is the most abundant material with which to fill this data gap. Here we report genetic analyses of fossil pollen retrieved from Holtjarnen postglacial lake sediment in Sweden and show that plastid DNA is recoverable from Scots Pine and Norway spruce pollen grains that are 100 and 10 000 years old. By sequencing clones from two short plastid PCR products and by using multiple controls we show that the ancient sequences were endogenous to the fossil grains. Comparison of ancient sequences and those obtained from an extant population of Scots pine establishes the first genetic link between extant and fossil samples in this species, providing genetic continuity through time. The finding of one common haplotype present in modern, 100-year old and 10 000-year old samples suggests that it may have persisted near Holtjarnen throughout the postglacial period. This retrieval of ancient DNA from pollen has major implications for plant palaeoecology in conifer species by allowing direct estimates of population dynamics in space and time.

Recent advances in molecular biology offer now excellent tools to obtain DNA information from well-preserved fossil material and the discovery that ancient DNA can be amplified by polymerase chain reaction (PCR) has added a direct temporal dimension to many evolutionary studies. By using a method we recently developed for the analysis of DNA in fossil pollen, we examined the chloroplast DNA sequence variation among fossil pollen grains of Scots pine (Pinus sylvestris L.) incorporated in a Holocene sediment core collected in central Sweden. DNA sequences data obtained from fossil (100 and 10,000 cal. BP) and modern samples revealed a number of different haplotypes and their relationships were visualised by a statistical parsimony network analysis. Results from this study establish a first genetic link between modern and fossil DNA samples and provide unique genetic information useful for understanding the origin and the evolution of Scots pine ancient populations.

The potential of ancient DNA research is far reaching in evolutionary studies and offers many opportunities, several of which have been little explored in plant palaeoecology. Recent genetic studies of fossil pollen of Scots pine retrieved from postglacial lake sediments in Sweden showed that chloroplast DNA can be recovered and used from remains as old as 10 ka. By sequencing and comparing short, yet very informative, chloroplast DNA regions from fossil and modern specimens we can now read the evolutionary message inscribed in this genome and learn how specific regions have evolved after the last glaciation. Can this approach be used also to study the evolutionary changes occurred both in time and space in plant populations during the last glaciation?

The chloroplast trnS-psbC gene regions from total genomic DNA of 119 accessions from seven small millet species were amplified by polymerase chain reaction (PCR) and digested with eight restriction enzymes individually as well as in combinations of two enzymes to

**Laboratory of Molecular Population Genetics, Department of Biology, Graduate School of Science, Kyushu University, Fukuoka 81 2-858 1, Japan ***Laboratory of Forest Genetics and Tree Breeding, PO Box. 238, Aristotle University of Thessaloniki, GR-54006 Thessaloniki

Abies nebrodensis (Lojac.) Mattei (Sicilian fir) is an endangered species represented by only one population of 29 adult individuals occurring in a limited area of the Madonie Range in northern Sicily (Italy). Taxonomic boundaries between this taxon and the neighboring Abies species are not clear. In this study, we used six chloroplast simple-sequence repeats (cpSSRs) to investigate the population genetic structure and the distribution of chloroplast haplotypic variation in A. nebrodensis and three of the neighboring Abies species: Abies alba (Mill.), Abies numidica (De Lann) and Abies cephalonica (Loud.). Our aims were to quantify the level of cpDNA differentiation within the Abies populations and to shed light on the history of A. nebrodensis. Diversity levels based on the haplotype frequency at six cpSSRs were high, especially in A. alba and A. cephalonica. In all, we found 122 haplotypes among the 169 individuals analyzed, and the four species were distinguished from each other by their haplotype composition. The majority of the haplotypes (76%) were detected only once, but in A. nebrodensis seven individuals (41% of the sample population) shared the same haplotype. Moreover, the seven A. nebrodensis individuals with an identical haplotype showed a tendency to be geographically grouped within the population’s limited range. The analysis of molecular variance (AMOVA) showed a significant difference in the level of apportionment of gene diversity between the species A. alba and A. cephalonica (F-ST=0.191 and 0.012, respectively). AMOVA analysis conducted over all populations from the four species showed that 19% of the total cpSSR variation was attributable to differences among species, 6% was due to differences among populations within species, and 74% to differences within populations. The high percentage of unique haplotypes identified confirms the power of cpSSR haplotype analysis for identifying individual trees in individual Abies populations. Our results in

We used PCR-RFLP analysis of the chloroplast DNA of the genus Abies (family Pinaceae), to determine if the method could be employed to detect inter-specific variation in this genus and to study how the variation was distributed in different regions of the genome. Ten different chloroplast DNA regions, consisting of coding and non-coding DNA sequences, were amplified with specific primers in ten different Abies taxa. The amplification products were digested with several restriction enzymes. The results showed that the chloroplast genome is highly variable in mast of the investigated taxa and contains multiple variable regions that appear to be distributed throughout the whole genome. Species-diagnostic markers were found for four of the ten investigated species. Unexpectedly, intra-specific variation was also detected in four species. It is likely that further studies, including larger sample sizes and/or more powerful methods for the detection of chloroplast DNA variation. will reveal additional variation for this genus.

PARDUCCI, L., SZMIDT, A. E., VILLANI, F., WANG, X.-R. and CHERUBINI, Abies alba in Italy. - Hereditas 125: 11 - 18. Lund, Sweden. ISSN 001 8-0661. Received February 28, 1996. Accepted August 26, 1996. Italy represents the southern limit of Abies alba (Mill.) (