Discussion
5S-IGS divergence and
paralogy/homeology
With the exception of few variants (‘Crenata A’, relict lineage
variants, partially deleted very short O-types), there is no evidence
for sequential decay. The relatively low GC contents (range: 33.2 –
45.1%) as compared to other Fagaceae such as oaks, appear to be
characteristic of nuclear spacers of Fagus , and its nucleome in
general (NCBI GenBank acc. no. QCXR00000000.1 and BKZX00000000.1; not
yet annotated, accessed on 25/11/2020). Thus, pseudogeny can be
largely ruled out as the cause for the detected 5S-IGS divergence.
Rather, the presence of two stable co-dominant main sequence clusters in
each sample (Fig. 6; Tables 2, 3) fits with the only available
cytogenetic data for Fagus (Ribeiro et al., 2011), showing two
paralogous, potentially functional 5S rDNA pericentromeric loci (and
four terminal 35S rDNA loci) in F. sylvatica . This is a rare
feature within Fagaceae, usually showing only one 5S rDNA locus
(www.plantrdnadatabase.com;
accessed 15/08/2020, Chokchaichamnankit & Anamthawat-Jonsson, 2015).
Additional or odd numbers of (unpaired) loci were found in single
individuals and attributed to hybridisation and auto-polyploidisation
(Chokchaichamnankit et al., 2008, Ribeiro et al., 2011). Comprehensive
data for comparisons with other families in the Fagales are currently
lacking, with the exception of Corylus (Betulaceae) showing a
single 5S locus and much lower intra-individual, intra-specific
divergence (Forest & Bruneau, 2000).
In F. japonica, the longer, GC-rich O-type and shorter, GC-normal
I-type, represent two highly divergent, only distantly related lineages;
their divergence (Table 1) parallels that between subgenera of oaks
(Denk & Grimm, 2010; Simeone et al., 2018; Piredda et al., 2020 for
first HTS data) and between genera in Betulaceae (Forest et al., 2005).
Both types are abundant: although HTS results cannot be generally
considered quantitative (Lamb et al., 2019), I- and O-type variants
appear co-dominant, while type X variants, representing a second
ingroup-related lineage of ambiguous phylogenetic affinity, are rarer
(Fig. 7; Supplementary file S1, section 4.3). This situation is similar
to the one reported for cloned ITS data: 35S rDNA-cistron ITS regions ofF. japonica and its sister species F. engleriana (mainland
China) and F. multinervis (Ullung Do, South Korea) show extreme
length and sequence heterogeneity with up to three divergent main
sequence types, while the ITS of the F. crenata-sylvatica lineage
is more homogeneous and poorly sorted (Denk et al., 2005; Grimm et al.,
2007).
In the 5S-IGS of the crenata-sylvatica lineage, the length and
sequence differences are less pronounced, with the longer A-type
variants being generally less derived and less abundant than the
shorter, much more abundant and more diverse B-type variants (Figs 3–5;
Supplementary file S1, appendix A). The divergence between these two
types is also comparable to intra-sectional diversity in oaks and
genus-level diversity in Betulaceae. The crenata-sylvaticasamples exhibit increasing dominance of type B over type A (Fig. 7):
weakly developed in Iranian F. orientalis , increased in European
samples following a (south)east/(north)west gradient (Greece – Italy –
Germany), and strongest in F. crenata (type A almost absent). In
addition, putative relicts (Fig. 5) can be found that lack/mix features
of A- and B-type variants (Supplementary file S4) or are clearly related
to the outgroup variants, type O (‘European O’; Figs 3, 4, 6). Hence, we
detected up to three major length classes referring to four principle
5S-IGS types of disparate phylogenetic affinity.
While two (or more) paralogous (and/or homeologous) 5S loci may have
facilitated ancient polymorphism (e.g. polyploidisation, hybridisation),
intragenomic silencing of homeologues leading to pseudogeny (reviewed in
Volkov et al., 2007; see Volkov et al., 2017 for a case of an ancient
allopolyploid) may cause the observed detection differences. The HTS
primers bind to the highly conserved 5S rDNA. If these are strongly
degraded, the intergenic spacers of such arrays will not be in our
sample. Compared to other studied Fagaceae (e.g. Denk & Grimm, 2010)
and ITS data (Denk et al., 2005), reducing spacer length by reducing
AT-dominated length-polymorphic regions may represent a general trend
(root-tip distances in Fig. 5 reflect sequence derivation). Within
beech, this trend is only obvious in the crenata-sylvaticalineage: the AT-richer A-types are slightly longer than the B-types
replacing them. In F. japonica, longer, GC-richer O-types are
rarer and less diverse than types I (Figs 6, 7; Supplementary files S1,
S2). Based on the high structural and sequence diversity,
counterbalanced by the large number of identical sequences detected in
each sample, a combined effect of both concerted and birth-and-death
evolution models must therefore be assumed for the 5S rRNA genes in
beech (Nei & Rooney, 2005; Galian et al., 2014). Thus, even if there
are two (or more) loci in all species of Fagus , they may not be
paralogous (in a strict sense) but rather act as homeologues, i.e. they
differ in position but not in function and are affected by inter-array
(inter-loci) recombination and limited concerted evolution.
Formation of 5S-IGS gene pools in the F. crenata – F.
sylvatica s.str.
lineage
Based on the ML results of the 686-tip tree, the individual samples, and
the 38 selected variants (Figs. 3, 5, 6), the potential disruption and
assembly of ancient common gene pools can be discussed using the NNet
network (Fig. 4). The closer two clusters (neighbourhoods, if defined by
an edge bundle) are in the network, the more recent is their split.
Mixed clusters typically comprise either shared ancestral variants or
variants propagated during (past) gene flow. Pronounced ‘trunks’ imply
genetic drift and isolation, while ‘fans’ reflect gradual radiation (see
also Hipp et al., 2020 for oaks). The distances across the graph
represent genetic distances, thus, the amount of genetic drift: the
fixation rate of new mutations and their propagation within the genome
and a population’s gene pool. Further, the network does not assume
dichotomy, which may be a poor model for the evolution and propagation
of 5S-IGS variants within a species’ gene pool. With currently available
methods, it is impossible to date potentially paralogous-homoeologous
multi-copy 5S IGS data. Considering the used taxon sample, method and
data, the divergence estimates by Renner et al. (2016) provide maximum
ages for lineage splitting in beeches. During the Eocene (56–33.9 Ma;
Cohen et al., 2013, updated), the lineages leading to F. japonicaand F. crenata + F. sylvatica s.str. started to diverge and
speciation in the crenata-sylvatica lineage probably started in
the late Oligocene (Chattian; 27.82–23.03 Ma). The fossil record
indicates that gene flow between the western and eastern populations of
the crenata-sylvatica lineage became impossible after the middle
Miocene, when the near-continuous northern Eurasian distribution of
beech forests became fragmented, at around 15–10 Ma (Denk, 2004;
Arkhipov et al., 2005). Gömöry et al. (2018) used allozymes and
approximate Bayesian computation to examine the demographic history of
western Eurasian beeches and suggested that F. sylvaticas.str. diverged from F. orientalis at ~1.2
Ma in the Pleistocene (Calabrian); this scenario is supported by the
leaf fossil record (e.g. Follieri, 1958). The diversity seen in IranianF. orientalis (Figs 3, 5) may well represent the original 5S-IGS
diversity within the western range of the Oligocene-Miocene precursor of
the crenata-sylvatica lineage. The poorly sorted ‘Western
Eurasian type B’ cluster is likely the direct result of a common origin
of the western Eurasian beeches and the Japanese F. crenata and
ongoing gene flow in the Miocene (relict ‘Crenata B0’; ‘Crenata B1’
grade; Fig. 3). It is possible that F. crenata (the
easternmost species) replaced its type A 5S-IGS variants with specific
‘Crenata type B2’ sequences. Originally, Fagus had a
(near)continuous range from Japan via central Asia to Europe, and it is
therefore possible that within this continuous area (extinct)
(sub)species of beech acquired 5S-IGS variants that survived within the
5S gene pool of populations like those observed in Iran. Beech forests
persisted throughout the entire Neogene in this area (and in Caucasus as
well) although experiencing severe bottlenecks (Shatilova et. al., 2011;
Dagtekin et al., 2020). The sharing of rare variants is consistent with
this scenario as they link isolated populations and otherwise distinct
species to a once common gene pool (e.g. ‘Relict’ lineage: ‘European O’,
‘Cross-Asia, Crenata B2’; Figs 3–5). These variants are not part of the
terminal subtrees, but instead reflect ancient, largely lost diversity
that predates the formation of the modern species and possibly even the
final split between ‘subgenus Engleriana’ (represented by F.
japonica ) and ‘subgenus Fagus’ lineages (crenata-sylvaticalineage).
In Europe, both A-type and B-type 5S-IGS arrays were secondarily sorted
and homogenized to a certain degree: ‘European A’ and ‘Western Eurasian
B’ clades include ± high proportion of shared (“ambiguous”) 5S-IGS
variants in contrast to the highly coherent ‘European B’ clade (Figs 3,
4). Unhindered gene flow lasted much longer between Greek F.
orientalis and F. sylvatica s.str. than the Iranian F.
orientalis and/or Japanese F. crenata . A side effect of the
higher genetic exchange between the western populations, a putatively
larger active population size and more dynamic history, is the retention
of ancient 5S-IGS variants, which are likely relicts from a past
diversification.
Inter-species relationships and status of Iranian F.
orientalis
Our data confirm the close relationship of F. crenata withF. sylvatica s.l. and the deep split between the two Japanese
species, belonging to different subgeneric lineages. Results also agree
with (i ) previous morphological (Denk, 1999a, b) and
population-scale isoenzyme and genetic studies (Gömöry & Paule, 2010;
Bijarpasi et al., 2020), which identified a split between disjunct
populations traditionally considered as F. orientalis in Europe
and adjacent Asia Minor (Iran and Caucasus); and (ii ) the
relatively recent contact and mixing between the westernmost F.
orientalis (here represented by a Greek sample) and F. sylvaticas.str. (Papageorgiou et al., 2008; Müller et al., 2019).
Considering all evidence, the Iranian F. orientalis must have
been isolated for a much longer time and likely deserves recognition as
distinct species. However, a formalisation requires comparative data of
populations in the Caucasus (including NE. Turkey; cf. Gömöry and Paule,
2010), where the holotype of F. orientalis comes from. According
to Denk (1999b), Gömöry & Paule (2010) and Gömöry et al. (2018), the
Caucasian populations are morphologically and genetically distinct from
both the western F. orientalis (including SE. Bulgarian and NW.
Turkish populations) and the Iranian populations. The high amount and
diversity of Iran-unique 5S-IGS A- and B-type variants, while being
geographically restricted, points to a complex history of the Iranian
beech. The Caucasian populations are geographically closer to the
Iranian, and they may have been in contact in the more recent past (via
Cis-Caucasia and Azerbaijan). Hence, some of the Iran-specific variants
may be shared by the Caucasian populations. Further genetic imprints in
the Iranian species could be a legacy from extinct beech populations
growing further east and the disjunct and taxonomically complex beeches
from the Nur (Amanos) Mts in south-central Turkey. The fact thatF. crenata and Iranian F. orientalis still carry
exclusively shared (‘Cross-Asia Crenata B2’) and related (‘Crenata A’;
‘Crenata B0’) 5S-IGS variants (Figs 3, 4) fits with a more or less
continuous range of beech populations from the southern Ural, via
Kazakhstan and Siberia, to the Far East until the end of the middle
Miocene (see above).
Potential of 5S-IGS HTS data to detect past and recent
reticulation
events
Beech has a complex biogeographic history in the Northern Hemisphere.
The crenata-sylvatica lineage represents the most evolved,
recently diverged branch (Supplementary file S4; Denk & Grimm 2009;
Renner et al., 2016). A reasonable model for beech evolution and
diversification in western Eurasia would include phases of range
contraction (isolation-speciation) and expansion (species mixing and
homogenisation) to explain the diffuse morphological evolution in
western Eurasian beeches. Such a scenario is supported by the observed
5S-IGS diversity (Fig. 8): speciation and isolation lead to the
accumulation of new lineage-specific variants, which are then exchanged
or propagated during episodes of favourable climate and massive
radiation of beech forests. Without data from Chinese and/or Taiwanese
species, it is impossible to assess whether the A- or B-types or the
partly pseudogenic relict type lineage(s) represent the original stock
of western Eurasian beeches and their Japanese sister species.
Nonetheless, since the A-type is largely lost in F. crenata , and
sequentially equally distant to the B-type than the F. japonicaingroup variants (I-type; Fig. 4), we postulate that both types are
present at least in some of the Chinese species and represent an ancient
polymorphism shared by all Eurasian ‘subgenus Fagus’ species.
Pronounced ancient nuclear polymorphism implies that at least some
modern beeches are of hybrid or allopolyploid origin. For the species of
‘subgenus Engleriana’ a hybrid origin would make sense, with theF. japonica ingroup variants representing the common ancestry
with the Eurasian clade of ‘subgenus Fagus’, while the outgroup variant
represents another, potentially extinct, lineage of high-latitude
beeches (Denk & Grimm, 2009; see also Fradkina et al., 2005; Grímsson
et al., 2016). A hybrid/allopolyploid origin would also explain the
extreme divergence observed in the ITS region of Fagus (Denk et
al., 2005) and appears to be supported by fossil evidence. Oldest
fossils unambiguously belonging to Fagus (dispersed pollen grains
from the early Paleocene of Greenland, with small size and narrow colpi
reaching almost to the poles; Grímsson et al., 2016) resemble ‘subgenus
Engleriana’. The subsequent radiation of beeches involved western North
America and East Asia, where fossil-taxa including morphological
characteristics of both modern subgenera are also known. For example,
the Eocene Fagus langevinii has branched cupule appendages as
exclusively found in extant Fagus engleriana along with ‘subgenus
Engleriana’ type pollen but resembles ‘subgenus Fagus’ in features of
leaf and nut morphology (Manchester & Dillhoff, 2005). Dispersed pollen
from the late Eocene of South China also resembles modern pollen of
‘subgenus Engleriana’ (Hoffman et al., 2019). This might reflect an
early phase in the evolution of beeches, during which the modern
subgenera were evolving, but not yet fully differentiated. By the early
Oligocene, fossil-species can clearly be assigned to either ‘subgenus
Fagus’ or ‘Engleriana’: F. pacifica from western North America
resembles ‘subgenus Fagus’ in leaf architecture and the cupule/nut
complex (Meyer & Manchester, 1997); cupules and leaves from the Russian
Far East can unambiguously be ascribed to ‘subgenus Engleriana’
(Pavlyutkin et al., 2014; as Fagus palaeojaponica , F.
evenensis , Fagus sp. 3).
Reticulation enriching the 5S-IGS pool is conceivable for the European
species as well. The first beeches arrived in Europe in the Oligocene
showing a general morphotype found across continental Eurasia (F.
castaneifolia ; Denk, 2004; Denk & Grimm, 2009). During the Miocene,F. castaneifolia was gradually replaced by F. haidingeri ,
the ancestor and precursor of all contemporary western Eurasian beeches
(Denk et al., 2005; Denk & Grimm, 2009). Like F. castaneifolia,
F. haidingeri shows high morphological plasticity. Hence, this
fossil-species may represent a species complex rather than a single
species. In addition, in southern Europe, gene flow between F.
haidingeri and another fossil-species, F. gussonii , might have
occurred. Our data confirm the potential for (sub)recent reticulation
and incomplete lineage sorting between and within the morphologically
distinct Greek F. orientalis and F. sylvaticas.str., and similar processes likely occurred repeatedly since
the Miocene. Notably, the Greek orientalis and F.
sylvatica s.str. comprise three main 5S-IGS lineages compared to only
two in the Iranian sample. In addition to the inherited polymorphism
shared with the Iranian F. orientalis (relatively similar type A,
‘Original A’ and ‘European A’; shared ‘Western Eurasian B’), we found a
highly abundant, moderately evolved but less diverse B-type (‘European
B’ in Figs 3–6), exclusive to Greek orientalis and F.
sylvatica s.str., likely reflecting the recent common origin of these
populations. In the context of F. gussonii, the second
fossil-species present in the Miocene of Europe (including Iceland) and
of uncertain affinity (Denk & Grimm, 2009), data from North American
extant species will be most valuable. Will they be clearly different (as
found for the ITS region and 2nd LEAFY intron;
Denk et al., 2005; Renner et al., 2016) or have they preserved
affinities with one of the here established lineages?