Aim: We have studied spatiotemporal genetic change in the Northern dragonhead, a plant species that has experienced a drastic population decline and habitat loss in Europe. We add a temporal perspective to the monitoring of dragonhead in Norway by genotyping herbarium specimens up to 200 years old. We also assess whether dragonhead has achieved its potential distribution in Norway. Location: Europe (mainly Norway)

Methods: We have applied a microfluidic array consisting of 96 SNP markers on 130 herbarium specimens collected from 1820 to 2008, mainly from Norway (83) but also beyond (47). We have compared our new genotype data with existing data from modern samples. We have modelled the species’ environmental niche and potential distribution in Norway using sample metadata and observational records.

Results: The SNP array successfully genotyped all included herbarium specimens. The captured genetic diversity was negatively correlated with distance from Norway. The historical-modern comparison revealed similar genetic structure and diversity across space and limited genetic change through time in Norway. The ENM suggests that dragonhead is anchored in warmer and drier habitats.

Main conclusions: With appropriate design procedures, the SNP array technology is promising for genotyping old herbarium specimens. We found no signs of any regional bottleneck. The regional areas in Norway have remained genetically divergent, however, both from each other and more so from populations outside of Norway, rendering continued protection of the species in Norway relevant. The ENM suggests that dragonhead has not fully achieved its potential distribution in Norway.

Specimens included

We generated SNP data from 130 herbarium specimens of Northern dragonhead, Dracocephalum ruyschiana, collected mainly in Norway, but also including 25 specimens from Sweden, 12 from Russia, four from Ukraine, and two from each of the countries Belarus, Switzerland and France. We reused SNP data from 355 modern Norwegian samples of dragonhead generated by Kyrkjeeide et al. (2020; 2022).

DNA extraction and genotyping

All manipulations of the herbarium specimens post-sampling were performed within the NTNU University Museum’s dedicated, positively pressurised paleo-genomics laboratory. About 0.5-cm2 of leaf material was removed from each herbarium specimen using a clean forceps and placed directly into a microfuge tube. The leaf maerial was pulverized with two Qiagen 3-mm tungsten carbide beads in a Qiagen TissueLyser LT at 50 Hz for 2 min. We extracted DNA using the DNeasy® Plant Mini Kit (Qiagen) with modifications from the manufacturer's instructions as described by Martin et al. (2014). We incubated the samples for 15 min at 37°C prior to spinning during the elution step. All extractions were performed using UV-sterilized equipment, and blank samples were always included to monitor for contamination. We measured DNA yield for 116 of the 130 herbarium samples using the Qubit dsDNA BR Assay Kit (Thermo Fisher Scientific, MA, USA) following the manufacturer's protocol. The DNA integrity was evaluated for the same 116 samples through agarose gel electrophoresis. The brightest band on the gel was regarded as an approximation for the sample's mean DNA fragment lenght. All samples were genotyped using the 96 x 96 SNP array developed by Kleven et al. (2019). The samples were genotyped on a Fluidigm EP1 instrument (Fluidigm Corporation, San Francisco, USA) according to the manufacturer’s protocol and scored using the Fluidigm SNP genotyping analysis software v4.5.1 (https://www.fluidigm.com/software). Positive and negative controls were included. We excluded SNPs with more than 10% missing data.

Datasets

We established two main datasets: (1) the “NOR” dataset of Norwegian samples only, which included SNP data from both the modern specimens, studied and generated by Kyrkjeeide et al. (2020, 2022; N=355), and the herbarium samples collected prior to 1950 (N=76), and (2) the “GLOB” dataset of all genotyped herbarium samples (83 Norwegian, 47 extra-Norwegian individuals).

References

Kleven O, Endrestøl A, Evju M, Stabbetorp OE, Westergaard KB (2019) SNP discovery in the northern dragonhead Dracocephalum ruyschiana. Conservation Genetics Resources https://doi.org/10.1007/s12686-018-1045-9

Kyrkjeeide MO, Westergaard KB, Kleven O, Evju M, Endrestøl A, Brandrud MK, Stabbetorp O (2020) Conserving on the edge: genetic variation and structure in northern populations of the endangered plant Dracocephalum ruyschiana L. (Lamiaceae). Conservation Genetics https://doi.org/10.1007/s10592-020-01281-7

Kyrkjeeide MO, Westergaard KB, Kleven O, Evju M, Endrestøl A, Brandrud MK & Stabbetorp O (2022) Conserving on the edge: genetic variation and structure in northern populations of the endangered plant Dracocephalum ruyschiana L. (Lamiaceae), Dryad, Dataset, https://doi.org/10.5061/dryad.j3tx95xgh

Martin MD, Zimmer EA, Olsen MT, Foote AD, Gilbert MTP, Brush GS (2014) Herbarium specimens reveal a historical shift in phylogeographic structure of common ragweed during native range disturbance. Molecular Ecology https://doi.org/10.1111/mec.12675

SNP code: 1=A, 2=C, 3=G, 4=T, 0=missing data