Introduction

Siberian forests are unique as they cover a vast area of about 263.2 million ha (Abaimov, 2010) dominated by a single genus of tree, the deciduous conifer larch (Larix Mill.). As the only extensive forest biome growing on continuous permafrost, it plays an important role for local communities and it provides critical ecosystem services in a global context including carbon stocks, climate feedbacks, permafrost stability, biodiversity and economic benefits (Herzschuh, 2019). It is therefore important to understand how the genus and individual larch species will respond to a warming climate.
Frequent natural hybridization between the larch species make it difficult to distinguish taxa and the number of accepted species is still under discussion (Abaimov, 2010). This is one of the reasons why there is still little known about the population dynamics of Siberian larch species and the question remains of whether there have been migrations of larches in the current postglacial.
Sedimentary ancient DNA (sed aDNA) from lakes can act as an archive of the past and has been demonstrated to be a valuable tool in the study of past vegetation history (Jørgensen et al., 2012; Parducci et al., 2017; Willerslev et al., 2003). Mostsed aDNA studies focus on organellar DNA, as the higher copy number of organelles per cell compared to the nucleus allow a higher chance of preservation. The metabarcoding approach (Taberlet, Coissac, Hajibabaei, & Rieseberg, 2012) applied to DNA extracted from sediments is the most common, robust and fast technique to study past vegetation (Alsos et al., 2018; Niemeyer, Epp, Stoof-Leichsenring, Pestryakova, & Herzschuh, 2017; Pansu et al., 2015). A very short, but highly variable DNA fragment is PCR-amplified out of the pool of DNA fragments and subsequently sequenced using high-throughput sequencing. However, the method is not suited to resolve population dynamics of single species, as metabarcoding markers used for ancient degraded samples must be very short while at the same time flanked by primers that are conserved across a larger taxonomic group. Therefore, their taxonomic resolution is, in most cases, insufficient to resolve closely related species (Taberlet et al. 2007), let alone show sub-specific variation.
Sequencing of the entire DNA extracted from ancient sediments, termed metagenomic shotgun sequencing, has been shown to provide information on the entire taxonomic composition of the sample (e.g., fungi, bacteria, archaea; Ahmed et al. 2018; Parducci et al. 2019). By sequencing complete DNA molecules, it is possible to authenticate ancient sequences versus modern contaminants by their specific post-mortem DNA damage patterns towards the ends of the molecules (Ginolhac, Rasmussen, Gilbert, Willerslev, & Orlando, 2011). As it is not restricted to a specific DNA fragment, it also allows the retrieval of many different loci belonging to single species provided they are sufficiently concentrated in the sample. Another advantage is that this method avoids bias introduced by PCR. A major drawback, however, is the immense sequencing effort that must be expended to achieve a sufficient overview of the DNA present in a sample. Most of the sequences retrieved from ancient environmental samples are not assignable to a specific taxon because available sequence databases are still limited, and most assigned sequences are not of eukaryotic origin (Ahmed et al., 2018; Pedersen et al., 2016). Especially in the case of DNA extracted from lake sediments, the ratio of sequences assigned to terrestrial plants to total DNA sequenced is expected to be extremely low (Parducci et al., 2019).
A way to overcome the limitations of shotgun sequencing is to enrich the DNA of the focal species in the samples via hybridization capture prior to sequencing. Here, short fragments of DNA of the species and target sites of interest are used as baits, to which the corresponding sites of interest in ancient DNA libraries are hybridized. This technique, originally developed for modern DNA has already been successfully applied to various ancient samples ranging from single specimens (Ávila-Arcos et al., 2011; Maricic, Whitten, & Pääbo, 2010) to cave sediments (Slon et al., 2017a) and permafrost samples (Murchie et al., 2019). To date, it has not been applied to DNA extracted from ancient lake sediments, which are especially challenging due to the high diversity of organisms living in the water and sediments and around the lakes. With the exception of metabarcoding analysis, most ancient DNA studies focus on mammals, mostly using mitochondrial DNA (Carpenter et al., 2013; Dabney et al., 2013; Enk et al., 2016). Plants have received limited attention in ancient DNA research (Parducci et al., 2017), and complete chloroplasts have not yet been targeted for hybridization capture of ancient DNA.
Here we apply shotgun sequencing and a hybridization capture approach toseda DNA samples from a small lake in the Taymyr region of north-eastern Siberia. The study site lies in the boundary zone of two larch species, Larix gmelinii and Larix sibirica(Abaimov, 2010), with hybridization occurring between the boundary populations (Abaimov, 2010; Polezhaeva, Lascoux, & Semerikov, 2010). It has been hypothesized for this region, that a natural invasion of L. gmelinii into the range of L. sibirica occurred during the Holocene (Semerikov, Semerikova, Polezhaeva, Kosintsev, & Lascoux, 2013). The lake is situated in the treeline ecotone with scattered patches of L. gmelinii occurring in the area (Klemm, Herzschuh, & Pestryakova, 2016). A sediment core of the lake has already been extensively studied using pollen analysis, DNA metabarcoding and mitochondrial variants (Epp et al., 2018; Klemm et al., 2016) making it an ideal site to study ancient larch population dynamics based on chloroplast DNA.
As a proof of concept, four samples were both shotgun sequenced and enriched for the chloroplast genome of L. gmelinii . We compare the proportion of classified reads at the domain level and the coverage of the Larix chloroplast genome of these methods. This study highlights the first successful recovery of near-complete chloroplast genomes from ancient lake sediments.