Sascha Liepelt

Wind-dispersed pollen mediates postglacial gene flow among refugia

A long-term genetic legacy of refugial isolation has been postulated and was demonstrated for maternal refugial lineages for numerous plant and animal species. The lineages were assumed to have remained separated from each other for several glacial periods. The conifer Abies alba Miller, silver fir, is an excellent model to test whether pollen-mediated gene flow may eliminate the genetic imprints of Pleistocene refugial isolation. Two DNA markers with contrasting modes of inheritance were applied to 100 populations covering the entire range of silver fir in Europe. The markers exhibited each two highly conserved alleles based on an insertion/deletion of 80 bp in the fourth intron of the mitochondrial nad5 gene and on a synonymous substitution in the chloroplast psbC gene. The geographical distribution of the maternally inherited mitochondrial variation supported the existence of at least two refugia with two recolonizing maternal lineages remaining largely separated throughout the range. The cline of the nad5 allele frequencies was much steeper than the one of the two psbC alleles. The psbC cline was as wide as the whole range of the species. Our results provide striking evidence that even a species with very long generation times and heavy pollen grains was able to establish a highly efficient pollen-mediated gene flow between refugia. Therefore we postulate that an exchange of genetic information between refugia by range-wide paternal introgression is possible in wind-pollinated plant species.

Tree endurance on the Tibetan Plateau marks the world’s highest known tree line of the Last Glacial Maximum

Because of heterogeneous topographies, high-mountain areas could harbor a significant pool of cryptic forest refugia (glacial microrefugia unrecognized by palaeodata), which, as a result of poor accessibility, have been largely overlooked. The juniper forests of the southern Tibetan Plateau, with one of the highest tree lines worldwide, are ideal for assessing the potential of high-mountain areas to harbor glacial refugia. Genetic evidence for Last Glacial Maximum (LGM) endurance of these microrefugia is presented using paternally inherited chloroplast markers. Five-hundred and ninety individuals from 102 populations of the Juniperus tibetica complex were sequenced at three polymorphic chloroplast regions. Significant interpopulation differentiation and phylogeographic structure were detected (G(ST) = 0.49, N(ST) = 0.72, N(ST) > G(ST), P < 0.01), indicating limited among-population gene flow. Of 62 haplotypes recovered, 40 were restricted to single populations. These private haplotypes and overall degrees of diversity were evenly spread among plateau and edge populations, strongly supporting the existence of LGM microrefugia throughout the present distribution range, partly well above 3500 m. These results mark the highest LGM tree lines known, illustrating the potential significance of high-mountain areas for glacial refugia. Furthermore, as the close vicinity of orographic rear-edge and leading-edge populations potentially allows gene flow, surviving populations could preserve the complete spectrum of rear-edge and leading-edge adaptations.

Molecular identification of individual oak and fir trees from maternal tissues of their fruits or seeds

The applicability of DNA markers to purely maternal tissues has been scarcely addressed in trees. We have focused on non-parenchymatic maternal tissues of the fruits and seeds of pedunculate oak (Quercus robur L.) and silver fir (Abies alba Mill.) and investigated whether they can be used for a direct molecular identification of the mother trees. Total DNA with sufficient quantity and quality was extracted from single woody pericarps of acorns as well as from single dry wings of silver fir seeds. The DNA was analysed by PCR at highly polymorphic microsatellite loci. A comparison of the multi-locus genotypes from pericarps and wings with those of the respective mother trees revealed absolute identity. Thus, mother trees could be identified by genotyping their fruits or seeds. The results demonstrate the applicability of DNA fingerprinting to woody and/or dry seed tissues without the destruction of embryos and endosperm or a significant contamination. Progress is now expected in dispersal biology as well as in forensics and forest management.

Contrasting geographical patterns of ancient and modern genetic lineages in Mediterranean Abies species

The diversification and distribution of Abies species throughout the Mediterranean region has led to a complex of species which are difficult to classify. An open question is whether these mainly allopatric taxa have exchanged genetic information via secondary contact. We studied the variation and geographic distribution of paternally inherited chloroplast DNA markers in nine Mediterranean Abies taxa. Markers with high and low mutation rates were applied in order to differentiate among a scenario of secondary genetic contact and a scenario of complete isolation after speciation. The observed molecular variation was analysed using statistical parsimony, geostatistics, and measures of population genetic differentiation. Ancient paternal lineages, represented by markers with low mutation rates, were shared among species. The central and widespread A. alba retained all ancient lineages whereas other species exhibited fewer, down to a single lineage. In contrast, modern lineages, depicted by markers with high mutation rates, were largely separated among species. The western Mediterranean A. pinsapo and A. numidica were clearly separated from each other and from the remaining Abies species. This indicates the absence of secondary contact. The same scenario applies to the eastern Mediterranean Abies species. An exception is the parapatric complex of A. alba, A. cephalonica, and their supposed hybrid A. borisii regis, which exhibited evidence of secondary contact.

Differentiating Groups of Abies Species With a Simple Molecular Marker

Abstract The unambiguous identification of closely related species is useful for many practical purposes in forest tree species. For example, international laws require timber identification and the control of the origin of forest reproductive material. In this paper, we present a mitochondrial DNA marker which can be used to differentiate among groups of fir species (Abies spp.). Eight Mediterranean and one North American fir species (used as reference) were analysed at the fourth intron of the NAD subunit 5 gene. A total of six different haplotypes was identified, one in the American Abies concolor, the other five in Mediterranean species. Two different haplotypes were found each in the widespread A. alba and in A. cephalonica, one haplotype being shared among the two species. A single species specific haplotype was found in the near-eastern A. cilicica. The two southwestern species A. pinsapo and A. numidica shared one haplotype. The fifth haplotype was shared by all remaining eastern Mediterranean firs, A. cephalonica, A. bornmuelleriana, A. equi-troiani, and A. nordmanniana. Differences in haplotype sequences were mainly due to large insertions/deletions. Agarose gel electrophoresis thus provides a fast, cheap and reliable diagnosis method for species or species group identification.

Monitoring Plant Drought Stress Response Using Terahertz Time-Domain Spectroscopy

A terahertz measurement setup precisely monitors changes in the water status of multiple plants in experiments under controlled environmental conditions. We present a novel measurement setup for monitoring changes in leaf water status using nondestructive terahertz time-domain spectroscopy (THz-TDS). Previous studies on a variety of plants showed the principal applicability of THz-TDS. In such setups, decreasing leaf water content directly correlates with increasing THz transmission. Our new system allows for continuous, nondestructive monitoring of the water status of multiple individual plants each at the same constant leaf position. It overcomes previous drawbacks, which were mainly due to the necessity of relocating the plants. Using needles of silver fir (Abies alba) seedlings as test subjects, we show that the transmission varies along the main axis of a single needle due to a variation in thickness. Therefore, the relocation of plants during the measuring period, which was necessary in the previous THz-TDS setups, should be avoided. Furthermore, we show a highly significant correlation between gravimetric water content and respective THz transmission. By monitoring the relative change in transmission, we were able to narrow down the permanent wilting point of the seedlings. Thus, we established groups of plants with well-defined levels of water stress that could not be detected visually. This opens up the possibility for a broad range of genetic and physiological experiments.

Transcriptome versus Genomic Microsatellite Markers: Highly Informative Multiplexes for Genotyping Abies alba Mill. and Congeneric Species

The availability of high-resolution, cost-effective polymorphic genetic markers displaying Mendelian inheritance is a prerequisite for fine-scale population genetic analyses as well as informed conservation and sustainable management. Silver fir (Abies alba Mill.) is a widespread European species of economic and ecological importance for which genetic markers are needed but difficult to develop, as in most conifer species. In this work, we introduce two sets of new multiplexed transcriptome-derived expressed sequence tag microsatellites (EST-simple sequence repeats (SSRs)) which we compare to a set of multiplexed genomic microsatellites (gSSRs). For both marker types, transferability was tested in 17 congeneric taxa. A total of 16 new EST-SSRs and two new gSSRs were developed. The EST-SSR multiplexes produced easily scorable amplification patterns that allow rapid and cost-effective genotyping at low-error rates, and include loci that display very low null allele frequencies. Generally, EST-SSRs displayed lower polymorphism and frequency of null alleles, but higher genetic differentiation among populations than gSSRs. Preliminary tests revealed that the EST-SSR markers are highly transferable and polymorphic across Abies species. This study also confirmed that SSRs can be successfully developed using next-generation sequencing technology also in large genome species such as conifers.

Evidence of divergent selection for drought and cold tolerance at landscape and local scales in Abies alba Mill. in the French Mediterranean Alps.

Understanding local adaptation in forest trees is currently a key research and societal priority. Geographically and ecologically marginal populations provide ideal case studies, because environmental stress along with reduced gene flow can facilitate the establishment of locally adapted populations. We sampled European silver fir (Abies alba Mill.) trees in the French Mediterranean Alps, along the margin of its distribution range, from pairs of high‐ and low‐elevation plots on four different mountains situated along a 170‐km east–west transect. The analysis of 267 SNP loci from 175 candidate genes suggested a neutral pattern of east–west isolation by distance among mountain sites. FST outlier tests revealed 16 SNPs that showed patterns of divergent selection. Plot climate was characterized using both in situ measurements and gridded data that revealed marked differences between and within mountains with different trends depending on the season. Association between allelic frequencies and bioclimatic variables revealed eight genes that contained candidate SNPs, of which two were also detected using FST outlier methods. All SNPs were associated with winter drought, and one of them showed strong evidence of selection with respect to elevation. QST–FST tests for fitness‐related traits measured in a common garden suggested adaptive divergence for the date of bud flush and for growth rate. Overall, our results suggest a complex adaptive picture for A. alba in the southern French Alps where, during the east‐to‐west Holocene recolonization, locally advantageous genetic variants established at both the landscape and local scales.

Differential Gene Expression Reveals Candidate Genes for Drought Stress Response in Abies alba (Pinaceae)

Increasing drought periods as a result of global climate change pose a threat to many tree species by possibly outpacing their adaptive capabilities. Revealing the genetic basis of drought stress response is therefore implemental for future conservation strategies and risk assessment. Access to informative genomic regions is however challenging, especially for conifers, partially due to their large genomes, which puts constraints on the feasibility of whole genome scans. Candidate genes offer a valuable tool to reduce the complexity of the analysis and the amount of sequencing work and costs. For this study we combined an improved drought stress phenotyping of needles via a novel terahertz water monitoring technique with Massive Analysis of cDNA Ends to identify candidate genes for drought stress response in European silver fir (Abies alba Mill.). A pooled cDNA library was constructed from the cotyledons of six drought stressed and six well-watered silver fir seedlings, respectively. Differential expression analyses of these libraries revealed 296 candidate genes for drought stress response in silver fir (247 up- and 49 down-regulated) of which a subset was validated by RT-qPCR of the twelve individual cotyledons. A majority of these genes code for currently uncharacterized proteins and hint on new genomic resources to be explored in conifers. Furthermore, we could show that some traditional reference genes from model plant species (GAPDH and eIF4A2) are not suitable for differential analysis and we propose a new reference gene, TPC1, for drought stress expression profiling in needles of conifer seedlings.

Within-range translocations and their consequences in European larch.

In contrast to biological invasions, translocations of individuals within a species range are understudied, due to difficulties in systematically detecting them. This results in limited knowledge about the corresponding processes and uncertainties regarding the status of extant populations. European larch, a forest tree whose fragmented native distribution is restricted to the Alps and to other Central European mountains, has been massively planted for at least 300 years. Here we focus on the genetic characterization of translocations having taken place within its native range. Microsatellite variation at 13 nuclear loci and sequence data of two mitochondrial DNA fragments were analyzed on the basis of a comprehensive range-wide population sample. Two complementary methods (Geneclass and Structure) were used to infer translocation events based on nuclear data whereas mitochondrial data were used for validation of these inferences. Using Geneclass, we found translocation events in a majority of populations. Additional cases of translocation and many instances of admixture were identified using Structure, thanks to the clear-cut ancestral genetic structure detected in this species. In particular, a strong divide between Alpine and Central European populations, also apparent at mitochondrial markers, helped uncover details on translocation events and related processes. Translocations and associated admixture events were found to be heterogeneously distributed across the species range, with a particularly high frequency in Central Europe. Furthermore, translocations frequently involved multiple geographic sources, some of which were over-represented. Our study illustrates the importance of range-wide investigations for tracing translocations back to their origins and for revealing some of their consequences. It provides some first clues for developing suitable conservation and management strategies.