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\begin{document}

\title{Pleistocene origin and colonization history of Lobelia columnaris Hook.
f. (Campanulaceae: Lobelioideae) across sky islands of West Central
Africa}



\author[1]{Miguel Perez Perez}%
\author[2]{Wen-Bin Yu}%
\affil[1]{Northern Arizona University - Flagstaff Mountain Campus}%
\affil[2]{Xishuangbanna Tropical Botanical Garden}%


\vspace{-1em}



  
  \date{May 25, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
We aimed to infer the phylogenetic relationships of populations of
Lobelia columnaris using chloroplast genomes and estimate the divergence
time to reconstruct its historical colonization on the sky islands of
Bioko and Cameroon. Specifically, we aim to answer the following
questions: (1) What is the phylogenetic relationship among Bioko Island
and Cameroon populations? (2) Are the older populations found on the
older sky islands? (3) Does the colonization history reflect the age of
the sky islands? We assembled novel plastomes from 20 individuals of L.
columnaris from five mountain systems. The plastome data was explored
with phylogenetic analyses using Maximum likelihood and Bayesian
Inference. The complete plastome size varied from 164,609 bp to 165,368
bp. The populations of L. columnaris have a monophyletic origin,
subdivided into three plastome-geographic clades. The plastid
phylogenomic results and age of the sky islands indicate that L.
columnaris colonized first along the Cameroon Volcanic Line's young sky
islands. The earliest divergent event (1.54 Ma) split the population in
South Bioko from those on the mainland and North Bioko. The population
of South Bioko was likely isolated during cold and dry conditions in
forest refugia. Presumably, the colonization history occurred during the
middle-late Pleistocene from South Bioko's young sky island to North
Bioko and the northern old sky islands in Cameroon. Furthermore, the
central depression with lowland forest between North and South Bioko is
a current geographic barrier that keeps separate the populations of
Bioko from each other and the mainland populations. The Pleistocene
climatic oscillations led to the divergence of the Cameroon and Bioko
populations into three clades. L. columnaris colonized the older sky
island in mainland Cameroon after establishing South Bioko's younger sky
islands. The biogeography history was an inverse progression concerning
the age of the Afromontane sky islands.%
\end{abstract}%



\sloppy


\textbf{Introduction}

The Pleistocene's climate and environmental oscillations caused
fragmentation and expansion of lowland tropical rain forests, mountain
forests, and savannas of Upper Guinea and Lower Guinea, Africa (Duminil
et al. 2015). These climate oscillations shaped Afro-alpine species
distribution, such as \emph{Lobelia giberroa,} which occurs on mountains
with altitudes between 3500 and 6000 m in East Africa (Kebede et al.
2007). Forest fragmentation during the Pleistocene was a driver that
gave origin to allopatric speciation of plants and animals. Likewise,
forest refugia and rivers contributed to current diversity patterns in
West Central Africa (Nicolas et al. 2012). Moreover, during the
Pliocene-Pleistocene epochs, there was substantial volcanic activity in
West Central African and East Africa, such as the Eastern Arc Mountains
of Tanzania and Kenya (Measey \& Tolley 2011).

The Cameroon Volcanic Line (CVL) is an 1800 km SW-NE topographical
feature that extends from the Gulf of Guinea to onshore central Cameroon
(Adams et al. 2015). This volcanic chain was an ancient forest refuge in
West Central Africa (Demenou et al. 2020). The CVL comprises plateaus
and 11 dormant volcanoes, and Mount Cameroon, a currently active volcano
(Chauvel et al. 2005). Only three volcanic peaks in the CVL have
elevations above 3000m (Pico Basil\selectlanguage{ngerman}é, Mt. Cameroon and Mt. Oku). The
geological structure of the CVL is a combination of tectonic and
volcanic origins with unequal ages ranging from the middle to late
Tertiary (Jesus et al. 2005). The oldest mountains are in the north,
with a trend of decreasing age of volcanic activity in the southern area
(Missoup et al. 2016). The marked geographical separation and isolation
of the mountains are analogous to islands in the sky or sky islands. Sky
islands are considered natural laboratories for studying evolutionary
patterns and processes that lead to the accumulation of diversity (Cox
et al. 2014). Moreover, the sky islands and sky island archipelagos of
the Gulf of Guinea and West Central Africa possess an extraordinary
diversity of angiosperms (Figueiredo 1994), small mammals (Missoup et
al. 2016), and amphibians (Zimkus \& Gvo\selectlanguage{polish}ž\selectlanguage{english}d\selectlanguage{ngerman}ík 2013). This African region
is part of the Guinea biodiversity hotspot (Myers et al. 2000) and is
critical for the conservation of endemic species of plants and animals
that inhabit the sky islands (Tropek \& Konvicka 2009).

Giant lobelias may have experienced rapid diversification in East Africa
mountains and subsequently dispersed to West Africa (Knox \& Li
2017).\emph{Lobelia columnaris} Hook f. (Campanulaceae: Lobelioideae) is
listed as a vulnerable species in the IUCN Red List of Threatened
Species 2015 (Cheek \& Thulin 2015). \emph{L} . \emph{columnaris}
and\emph{L} . \emph{barnsii} Exell (Mabberley 1974a) are the two giant
lobelia species known from the tropical West Central Africa and Gulf of
Guinea. \emph{L} . \emph{columnaris} is endemic to Bioko's mountains and
highlands (Equatorial Guinea), Nigeria, and Cameroon. This giant lobelia
grows in discontinuous populations between 1000m to 3000m in different
ecological habitats including submontane grasslands, forest clearings
transformed into grasslands overgrown with \emph{Pteridium
aquilinum}(L.) Kuhn\emph{,} and along streams and subalpine meadows.

Little is known about the origin and colonization histories of
angiosperms in Bioko and Cameroon. However, a few phylogeographic
studies have documented the lineages of plants and animals currently
present in Bioko and Cameroon. For example, the Afromontane
genus\emph{Lynchis} had several dispersals from Ethiopia and the Western
Rift Mountains and recently dispersed to West Africa (Popp et al. 2007).
The hypothesis of recurrent connections over time between the West and
East African mountains provides a framework to discuss the
biogeographical origin among close relatives in both bioregions, like
the endangered and endemic Mount Oku rat, \emph{Lamottemys okuensis} in
the CVL (Missoup et al. 2016). However, some taxa have a geographically
widespread distribution, from the eastern mainland to the outlying
islands of western Africa, like the endangered \emph{Prunus africana}
(Dawson \& Powell 1999).

The present study's objective is to infer the phylogenetic relationships
of populations of \emph{L. columnaris} using chloroplast genomes and
estimate the divergence time to reconstruct its historical colonization
on the sky islands of Bioko and Cameroon. Specifically, we aim to answer
the following questions: (1) What is the phylogenetic relationship among
Bioko Island and Cameroon populations? (2) Are the older populations
found on the older sky islands? (3) Does the colonization history
reflect the age of the sky islands?

\textbf{Materials \& Methods}

Study area

Bioko Island lies on the continental shelf and is separated from the
Cameroon coast by 32 km of shallow water (60m). Bioko was separated from
mainland Africa 10, 000 years ago by the rise of sea level at the end of
the last glacial period (Schabetsberger et al. 2004).

A recent study of the geochemistry of volcanic rocks from Bioko dates
the main three strato-volcanoes at \textless{}1.3±0.07 Ma; K/Ar
(Yamgouot et al. 2016). This dating indicates that the sky islands of
Bioko (South and North) are very young without difference in age.
Furthermore, a central depression with a maximum elevation of 500m
separates Pico Basilé (3011m) in the North and the Pico Biao-Moka
(2009m) and the Gran Caldera (2260 m) in the South (Schabetsberger et
al. 2004). The vegetation of Bioko is arranged in elevational rings
dominated by Guineo-Congolian tropical species with Afromontane elements
appearing at higher elevations (Fa et al. 2000).

Mainland Cameroon comprises several Afromontane sky islands. In the
southwest Mount (Mt.) Cameroon is the highest volcano (4085m) of West
Central Africa. In the northeast, Mount Oku is another high peak with an
elevation of 3011m. Likewise, Bamenda Plateau and Bambutos Mountains
represent the orography of the North region. Mountains from the North
Central, such as Mt. Oku and Bamenda-Banso highlands (2260m), uplifted
during the Cenozoic (Oligocene to Miocene) (Missoup et al. 2016).
Mainland southern volcanoes like Mt. Cameroon are the youngest with
origins during the Pliocene to Pleistocene (Zimkus \& Gvo\selectlanguage{polish}ž\selectlanguage{english}d\selectlanguage{ngerman}ík 2013).

The vegetation is arranged in elevational bands within the montane
forest and has been highly disturbed by grazing, fire, and human
activities, except on Mt. Cameroon (Ineich et al. 2015).

Study plants, DNA extraction and sequencing

\emph{Lobelia columnaris} is a semelparous herb or shrub up to 1 m, with
curved purple corollas organized on a terminal or multi-terminal
panicle. The protandrous flowers are present in the dry season and live
approximately ten days (Bartos et al. 2012). The flowers produce sugared
nectar that attracts birds such as the Orange-tufted sunbird
(\emph{Cinnyris bouvieri} ) (Janecek et al. 2012; Riegert et al. 2011)
and Hymenoptera insects. The flowers are considered bee-pollinated
(Givnish et al. 2009). The fruit is a capsule with thousands of winged
seeds (Mabberley 1975) dispersed by wind (Knox \& Palmer 1998; Mabberley
1975).

Fieldwork was conducted in February and March of 2015 with permits from
Universidad Nacional de Guinea Ecuatorial, Republic of Equatorial
Guinea, and from the Ministry of Scientific Research and Innovation,
Republic of Cameroon. Locality and collection data on the samples
of\emph{L. columnaris} used in this study are given in Table 1. Voucher
specimens are deposited in the Philadelphia Herbarium (PH). Voucher
duplicates were deposited in the National Herbarium of Cameroon (YA) and
Flora of Guinea-Real Jardín Botánico Madrid (MA).

Table 1. Locality and collection data of \emph{Lobelia columnaris}\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
Region & Population (\# ind) & Sky Island & Elevation (m)\tabularnewline
\midrule
\endhead
North Bioko & 3104 (2) & Pico Basil\selectlanguage{ngerman}é & 2947\tabularnewline
North Bioko & 3105 & Pico Basil\selectlanguage{ngerman}é & 2836\tabularnewline
North Bioko & 3106 (2) & Pico Basil\selectlanguage{ngerman}é & 2322\tabularnewline
South Bioko & 3107 (2) & Gran Caldera & 1001\tabularnewline
South Bioko & 3108 & Pico Biao-Moka & 1582\tabularnewline
South Bioko & 3109 & Pico Biao-Moka & 1539\tabularnewline
South Bioko & 3110 (2) & Pico Biao-Moka & 1157\tabularnewline
Cameroon & 3111 & Mt. Oku & 2381\tabularnewline
Cameroon & 3112 (2) & Mt. Oku & 2522\tabularnewline
Cameroon & 3113 & Mt. Oku & 2197\tabularnewline
Cameroon & 3114 & Mt. Oku & 1977\tabularnewline
Cameroon & 3116 & Mt. Oku & 2407\tabularnewline
Cameroon & 3117 & Mt. Bamenda & 1798\tabularnewline
Cameroon & 3118 (2) & Mt. Bamenda & 1723\tabularnewline
\bottomrule
\end{longtable}

DNA was extracted from silica-dried leaf samples of 20 individuals
of\emph{L. columnaris} using a modified cetyltrimethylammonium bromide
(CTAB) protocol (Doyle \& Doyle 1987). Library construction, sequence
generation, and bioinformatics processing were done at the Indiana
University Center for Genomics and Bioinformatics. Sequence generation
was performed using Illumina NextSeq and Illumina MiSeq platforms.
Additionally, we downloaded 22 plastome sequences of 22 taxa of
Campanulaceae from TreeBASE no. S15797 (Knox 2014) and GenBank
accessions (Knox \& Li 2017).

A total of 20 individual plastid genomes of \emph{L. columnaris} were
newly sequenced and assembled. Eleven plastomes represent populations
from three sky islands in Bioko (Equatorial Guinea), and nine plastid
genomes are from two sky islands in Cameroon (Figure 1). Moreover, the
phylogenetic tree included two more plastome sequences of \emph{L.
columnaris} from South Bioko (Perez Perez 3103, MF061188) and another
from Mt. Cameroon (Muasya 2085, MF061187) obtained from GenBank (Knox \&
Li 2017) .\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

Figure 1. Geographic distribution of plastomes of \emph{Lobelia
columnaris.}

The total dataset included 44 plastomes (22 of \emph{L. columnaris} and
22 from other Campanulaceae taxa).

Assembling and annotation of the chloroplast genomes

The raw paired-end reads were filtered and \emph{de novo} assembled
using the GetOrganelle toolkit (Jin et al. 2020). The filtered reads
were assembled using the SPAdes version 3.9 using \emph{k-mer} 21, 33,
45, 65 and 85 (Bankevich et al. 2012). To retain pure chloroplast
contigs, the final ``fastg'' files were filtered using the ``slim''
script of GetOrganelle toolkit. The filtered De Brujin graph was viewed
and the final sequence exported using Bandage (Wick et al. 2015). The
chloroplast genome was automatically annotated using CpGAVAS (Liu et al.
2012), then adjusted using Geneious version 9.0 (Kearse et al. 2012).

Phylogenetic analyses

The whole chloroplast genome matrix was aligned using MAFFT version 7.1
(Yamada et al. 2016). Only one inverted repeat region was used in the
phylogenetic analyses. Two matrixes were prepared, one including all
gaps, and another removed gaps using trimAl (Capella-Guti\selectlanguage{ngerman}érrez et al.
2009) by ``-gt 0.4 -st 0.001 -cons 40''. Maximum Likelihood (ML) and
Bayesian Inference (BI) methods were used to reconstruct phylogenetic
trees. No nucleotide positions were excluded from analyses. The ML tree
analyses and bootstrap estimation of clade support were conducted with
RAxML version 8.2.10 (Stamatakis et al. 2008). These analyses used the
GTR substitution model with gamma-distributed rate heterogeneity among
sites and the proportion of invariable sites estimated from the data.
Support values for the node and clade were calculated from 1000
bootstrap replicates. Bootstrap support (BS) \selectlanguage{english}[?] 70 is considered as well
supported (Hillis \& Bull 1993). The BI analyses were performed using
MrBayes version 3.2.6 (Ronquist \& Huelsenbeck 2003), with DNA
substitution models selected for each gene partition by the Bayesian
information criterion (BIC) using jModeltest version 2.1.10 (Darriba et
al. 2012; Guindon \& Gascuel 2003). Markov Chain Monte Carlo (MCMC)
analyses were run in MrBayes for 10,000,000 generations, with two
simultaneous runs, and each run comprising four incrementally heated
chains. The BI analyses were started with a random tree and sampled
every 1000 generations. The number of generations for the three datasets
was sufficient because the average standard deviation of split
frequencies for the datasets was lower than 0.005, and Potential Scale
Reduction Factor (PSRF) of Convergence Diagnostic (Gelman \& Rubin 1992)
for the datasets was 1.00. The first 25\% of the trees were discarded as
burn-in, and the remaining trees were used to generate a majority-rule
consensus tree. Posterior probability values (PP) \selectlanguage{english}[?] 0.95 were considered
as well supported (Alfaro et al. 2003; Erixon et al. 2003; Kolaczkowski
\& Thornton 2007). Both ML and BI analyses, as well as jModeltest, were
performed at the CIPRES Science Gateway (http://www.phylo.org).

Estimation of divergent times and phylogeographical history

Dating analyses were conducted using Markov Chain Monte Carlo (MCMC)
methods in BEAST version 2.4 (Bouckaert et al. 2014), which was
performed at the CIPRES Science Gateway (http://www.phylo.org). The
setting parameters in BEAUti, we chose ``BEAST model test'' for ``Site
model'', ``Relaxed Clock Log Normal'' for ``Clock model'', and ``Yule
Model'' for speciation. Meanwhile, we selected two crown nodes for
calibrations from published data using the CladeAge package (Matschiner
et al. 2016). (1) The \emph{Lobelia} \emph{thuliniana} \emph{-- L.
columnaris} clade was 8.0 Mya (sigma 1.0, offset: 0.0), and
(2)\emph{Lobelia} laxiflora - \emph{L. columnaris} clade was 21 Mya
(sigma 2.0, offset: 0.0) (Chen et al. 2016; Knox 2014). For
reconstructing the phylogeographical history of \emph{L. columnaris} , a
plastome phylogeny of the \emph{L.} \emph{thuliniana} \emph{-- L.
columnaris} clade including 22 samples of \emph{L. columnaris} and five
outgroups was estimated in BEAST2 with ``beast-classic package'' of
BEAUti. For each dataset, MCMC ran 200,000,000 generations and sampled
every 20,000 generations. The first 5,000 generations were removed as
``Pre Burnin''. Log output of the BEAST analysis was evaluated using
Tracer version 1.6. Effective sample sizes (ESS) of all parameters were
more than 200, indicating that the estimations were confident. Maximum
clade credibility (MCC) tree was generated using TreeAnnotator by
setting ``Mean heights'' for the ``Node heights''. The MCC tree was
visualized using FigTree version 1.4.2
(http://tree.bio.ed.ac.uk/software/figtree/).

\textbf{Results}

Characteristics of plastomes

The plastome of \emph{L. columnaris} is circular and quadripartite by
having a large single copy (LSC), a small single copy (SSC), and two
inverted repeated (IR) regions. The complete plastome size of varied
from 164,609 bp to 165,368 bp, with LSC from 82,421 bp to 82,904 bp, SSC
from 7,950 bp to 8025 bp, and IRs from 37,028 bp to 37636 bp (Figure 2).
The GC content of the whole plastome was 39.2 \%. The plastome contained
117 unique genes, including 80 recognized and three predicted
protein-coding regions (CDS), 30 tRNA, and 4 ribosomal RNA (rRNA) genes.
The IR regions had 14 CDS, seven tRNA, and four rRNA genes.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

Figure 2. Map of newly sequenced plastome of \emph{Lobelia columnaris} .

Phylogenetic relationships

Our phylogenetic results using maximum likelihood (ML) and Bayesian
inference (BI) generated identical tree topologies. We recovered a
monophyletic relationship among the populations of \emph{L.
columnaris}from Bioko and Cameroon. However, we distinguished three
highly supported clades in the phylogenetic tree. A common ancestor
closely relates the mainland Cameroon clades (Mt. Cameroon, Bamenda, and
Mt. Oku) with North Bioko (Pico Basil\selectlanguage{ngerman}é) sister to these clades. The
clade of southern Bioko (Pico Biao-Moka and Gran Caldera) is distinct
and sister to the previous subclades (Figure 3).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

Figure 3. Phylogenomic tree of \emph{Lobelia columnaris} .

Reconstruction of the phylogeographical history

The geographic distribution of the plastid genomes shows the distant
relationship between the populations from South Bioko and the other
populations in this study. In contrast, populations in the three sky
islands of mainland Cameroon are more closely related. Each pie graph
shows the proportion of plastome sequences from each geographic sky
island system (Figure 4).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

Figure 4. Geographic and phylogenomic relationship of plastomes of

\emph{Lobelia columnaris.}

Events of divergence times

Three important divergent events are noticeable on the genomic-dating
tree. The first event is dated 5.58 Ma, marking the divergence between
the ancestor of \emph{L. columnaris} and giant lobelias from East Africa
(Figure 5). The second occurred approximately 1.54 Ma and marked the
separation of populations in West Central Africa and North Bioko from
South Bioko. Finally, the third event is estimated to be 0.99 Ma
displaying the split of North Bioko and mainland Cameroon populations.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

Figure 5. MCC tree with 95\% HPD confident intervals for phylogenomic
relationships and estimation of divergence times obtained in BEAST.

\textbf{Discussion\emph{Phylogenetic relationship of populations}}

The ancestor of \emph{L. columnaris} spread from East Africa via the
Congo's Basin to establish in West-Central Africa approximately 5.5 Ma
(Knox \& Li 2017). The genealogical relationships among populations
of\emph{L. columnaris} in the present day were resolved with the
analyses of plastomes. However, populations within South Bioko's
subclade do not have high bootstrap values or posterior probabilities,
possibly because these populations diverged recently, and the plastome
may not be the right marker to show a robust phylogenetic resolution at
this fine scale.

We hypothesized that the populations on Bioko shared the same common
ancestor. However, our results refute the hypothesis. Our outcome
clearly shows an early colonization event in South Bioko. A second
divergence separated the populations of North Bioko and Cameroon.
According to this result, Bioko's two populations are not a monophyletic
group suggesting that the evolutionary history of the island populations
is more complex.

We found a phylogeographic pattern with three clades distributed on the
six sky islands. Remarkably, the two clades located in Bioko do not
share a more recent common ancestor. The third clade in Cameroon shares
a more recent common ancestor with populations in North Bioko.

Historical colonization

Sky island's age

The Hawaiian archipelago is well documented for the correlation between
age of the islands and their colonization and radiation of various
plants' lineages, such as the Hawaiian lobeliads (Givnish et al. 2009).
In contrast, the West-Central Africa sky archipelago does not have a
simple chronological age from east to west (Suh et al. 2008).
Nevertheless, the six sky islands that we studied have a clear
progression. Their estimated ages run from old (West-Central Cameroon)
to young mountains (Bioko and Mt. Cameroon). Our plastome
phylogeographic interpretation indicated that \emph{L.
columnaris}colonized the older sky islands in mainland Cameroon after
establishing South Bioko's younger sky islands. Moreover, the South
Bioko colonization predated North Bioko.

Overall, this result contradicts our sky island age hypothesis because
Cameroon's sky islands with estimated ages of 3.0, 22.0, and 31.0 Ma are
older than Bioko's sky islands (ca. 1.3 Ma) (Table 2). Our results
suggest a biogeography history with an inverse progression concerning
the age of the Afromontane sky islands.

Table 2. Elevation and estimated age of six sky islands of the Cameroon
Volcanic Line\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
\textbf{Sky Island} & \textbf{Elevation (m)} & \textbf{Age (Myr)} &
\textbf{Reference}\tabularnewline
\midrule
\endhead
Pico Biao-Moka & 2009 & 1.3 & Yamgouot et al., 2016\tabularnewline
Gran Caldera & 2260 & 1.3 & Yamgouot et al., 2016\tabularnewline
Pico Basil\selectlanguage{ngerman}é & 3011 & 1.3 & Yamgouot et al., 2016\tabularnewline
Mt. Cameroon & 4085 & 3 & Suh et al., 2008\tabularnewline
Mt. Bamenda & 2621 & 22-21 & Zimkus \& Gvozdik, 2013\tabularnewline
Mt. Oku & 3011 & 31-22 & Suh et al., 2008\tabularnewline
\bottomrule
\end{longtable}

Perhaps, South Bioko populations are in a basal position on the
ML/BI/Beast trees because the plastomes provide a skewed view of the
biogeographic history of \emph{L. columnaris} , or there is sampling
error because we sequenced only 1-2 individuals per populations.
Furthermore, we do not have population data from Nigeria, and we did not
find more populations in Cameroon, possibly for the local extinction of
populations caused by the intense human transformation of Afromontane
habitat on the sky islands.

Population differentiation

Microevolutionary processes could be an alternative explanation for the
colonization history of \emph{L. columnaris} in the Afromontane sky
islands of West Central Africa. The early split in \emph{L.
columnaris}populations between the mainland and South Bioko possibly
occurred via a founder event and genetic bottleneck or subsequent
genetic drift so that South Bioko plastomes are very distinct relative
to the other plastome groups. Furthermore, the two early splits (Figure
3) suggest two independent colonization events of \emph{L. columnaris}
on the island. Under this scenario, the colonization history was likely
from mainland Cameroon's old sky islands to the southern young sky
islands in Bioko.

This explanation should be developed with population genetic analyses to
explore the correlation between geographic distance and genetic
differentiation of populations and find which evolutionary forces are
responsible for population differentiation in Bioko.

Missing links

The lack of Nigerian populations of \emph{L. columnaris} in the
phylogenomic reconstruction likely is causing missing links between the
island and mainland populations. Future analyses with samples from
Nigeria will provide a better interpretation of colonization history.

Afromontane forest expansion and contraction

Eastern giant lobelias evolved in Afromontane forests at elevation
ranges from 1000 to 2500m. Through frost tolerant adaptations, some taxa
colonized the inhospitable Afroalpine elevations from 3000 to 5000m
(Hedberg 1969). We found that middle elevations are the most likely
habitat for \emph{L. columnaris} . Only Pico Basil\selectlanguage{ngerman}é's (North Bioko)
population extends its range from middle elevation up to
\textgreater{}3000m. At this altitude, the habitat is composed of shrubs
and subalpine meadows.

The paleoclimatic scenarios of the Afromontane vegetation during the
Pleistocene in West Central Africa are not well known. However, it is
known that climatic fluctuations changed the past vegetation patterns
dramatically. With pollen analysis is possible to reconstruct historical
processes (Kadu et al. 2011) like the presence of forest bridges that
connected sky islands and highlands (Kebede et al. 2007) and, as a
result, facilitated periodic expansions of the Afromontane forest
through lowland elevations (Mairal et al. 2017). This dynamic allowed
species to disperse and to be isolated in adjacent mountains (Zimkus \&
Gvo\selectlanguage{polish}ž\selectlanguage{english}d\selectlanguage{ngerman}ík 2013).

Under this scenario, \emph{L. columnaris} probably had changes in
population size during the Pleistocene, mainly caused by repeated
climatic oscillations and Afromontane fragmentation. Also, environmental
fluctuations caused the constriction of populations into forest refugia.
Later, populations expanded during warm and humid environmental
alternations (Gao et al. 2015). Therefore, the current disjunction
of\emph{L. columnaris} in sky islands probably was a Pleistocene product
of retraction and isolation of Afromontane forest and continuous
post-Pleistocene dispersal to empty niches.

Although not explored in the study, active volcanism and land use have
to be considered in a holistic view of the current distribution of this
giant lobelia.

Pleistocene refugia

The entire Cameroon line is considered a Pleistocene forest refuge for
its distinctive flora. Forest refugia are also supported because of the
high genetic diversity detected in different trees (Pineiro et al. 2017)
such as the genus \emph{Greenwayodendron} (Migliore et al. 2018), and
other flowering plants. For example, \emph{Arabis alpina} (L.) survived
the Pleistocene oscillations of temperature and drought in refugia. Once
the environmental conditions changed during the interglacial
periods,\emph{A. alpina} colonized or recolonized new sky islands in
East-Central Africa (Assefa et al. 2007).

It is possible that the populations of \emph{L. columnaris} were
dynamically isolated and expanded within and between the sky islands of
West Central Africa. However, Pico Biao-Moka in South Bioko might be a
Pleistocene forest refuge to Afromontane plant species.

Geographical barriers

The forest contraction that occurred during the Pleistocene radically
changed the landscape. Additional landscape changes occurred in the
region at the end of the last glacial period (ca. 10,000 years ago) when
a rise in sea level isolated Bioko from the African mainland (Jones
1994). A shallow channel separates Bioko from the Cameroon coast by 32
km (Schabetsberger et al. 2004). We do not have evidence of recent
dispersal and gene flow between North Bioko and mainland Cameroon.

The central depression with lowland forest (0-500 masl) between North
and South Bioko (Schabetsberger et al. 2004) exemplifies another
geographic barrier that probably maintains reproductive isolation
between the populations of \emph{L. columnaris} in Bioko Island. The
central depression consists of lowland forest vegetation.

The expansion, colonization, and recolonization of \emph{L.
columnaris}was possible by wind dispersal. Wind played an essential role
in dispersing tiny seeds altitudinally and latitudinally across sky
islands. In Cameroon, high ridges act as a natural forest corridor
connecting sky islands, and facilitating dispersal and gene flow among
contemporaneous populations (Smith et al. 2000).

Ecology and conservation

The type of ecological habitat may have a possible effect on the
morphology of \emph{L. columnaris} (Mabberley 1974b). This observation
has to be developed in future studies. Our study observed that
populations grow in a mosaic of ecological habitats, latitudinal, and
elevational gradients. Indeed, we observed phenotypic variation in some
traits, for example, in plant height, size of the inflorescence, leaves,
and flower colour. \emph{L. columnaris} is smaller in height,
inflorescence, and leaf length at a higher elevation (approx. 3000m) and
lower elevation (1000m). Elevations between 2000 to 2600m, the variation
is spectacular with greater height, inflorescence number, colour, and
leaf length.

The populations of \emph{L. columnaris} in mainland Cameroon are at high
risk of local extirpation because of excessive anthropogenic pressure on
montane forest fragments. Only for the populations on Mt. Cameroon,
which is a National Park, have conservation protection plans. The
scenario in Bioko for this giant lobelia and the Afromontane forest is
better. Bioko has an active conservation procedure in two of the three
sky islands (Müller \& Pócs 2007). Moreover, South Bioko is undisturbed
because of low human population density and supports the highest numbers
of plant and animal species on the island (see Jones 1994).

Study limits

Our results are limited by several factors. This preliminary study was
conducted with few individuals (1-2) for each population, and as such
should be considered a baseline for future studies. The available sample
sizes for every population is ten individuals. We hope to conduct future
studies with all the individual samples.

Social instability was the main reason no samples of \emph{L.
columnaris} were collected in Nigeria. Future fieldwork in Nigeria to
find and collect more populations will expand the sampling effort and
allow a more robust phylogeographic study. Populations from Mt. Cameroon
were under-collected because it was challenging to get the permits to
work in the National Park. Finally, this study presents a partial
history with the analysis of a non-recombinant marker. A more robust
analysis should include nuclear genomic sequences.

\textbf{Conclusions}

The plastome analysis provided an informative phylogenetic
reconstruction but partial colonization reconstruction of \emph{Lobelia
columnaris} . Overall, three groups are in West-Central Africa.

According to our phylogenomic calibration, the ancestor of \emph{L.
columnaris} arrived in West Central Africa (ca. 1.5 Ma) during the
Cameroon line's youngest volcanoes' uplift. The Pleistocene climatic
oscillations led to the divergence of the Cameroon and Bioko populations
into three clades. Likely, South Bioko was forest refugia during the
interglacial periods. Here, we show that the biogeographic history
of\emph{L. columnaris} does not follow the progression of ages of our
sky islands.

In more recent times, Bioko's central depression likely functions as a
geographic barrier to further isolate the population groups discovered
in our analysis (South Bioko versus North Bioko-Cameroon). Moreover,
grazing, burning, and deforestation are threatening Afromontane forest
patches with \emph{L. columnaris} in mainland Cameroon.

\textbf{Data accessibility} We are working the data storage from the
plastomes generated in this study. Data will be deposited in GeneBank

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\includegraphics[width=0.70\columnwidth]{figures/3-IGREZ-1-Fig-3-Phylogenetic-ML-results-among-populations-of-Lobelia-columnaris--1-/3-IGREZ-1-Fig-3-Phylogenetic-ML-results-among-populations-of-Lobelia-columnaris--1-}

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