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.