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Botanical Journal of the Linnean Society, 2023, XX, 1–12. With 3 figures.
Comparative phylogeography of a restricted and a
widespread heather: genetic evidence of multiple
independent introductions of Erica mackayana into
Ireland from northern Spain
JAIMEFAGÚNDEZ1,2,*, and PILARDÍAZ-TAPIA1,3
1Universidade da Coruña, BIOCOST research group, Centro Interdisciplinar de Química e Bioloxía
(CICA), Rúa As Carballeiras, 15071, A Coruña, Spain
2Universidade da Coruña, Departamento de Bioloxía, Facultade de Ciencias, 15071, A Coruña, Spain
3Instituto Español de Oceanografía (IEO-CSIC), Centro Oceanográfico de A Coruña, Paseo Marítimo
Alcalde Francisco Vázquez, 10, 15001, A Coruña, Spain
Received 2 May 2022; revised 24 October 2022; accepted for publication 15 November 2022
Species of flora and fauna occurring in the west of Ireland and south-west Europe, known as Lusitanian elements,
constitute a puzzling case of isolated populations of uncertain origin. Here we studied the population genetic
structure of the heather Erica mackayana in Ireland and northern Spain and compared it with its widespread close
relative Erica tetralix using single nucleotide polymorphisms (SNPs). We reconstructed phylogenetic relationships
using maximum likelihood (ML), inferred population genetic structure using cluster assignment and principal
component analysis, and estimated population genetic diversity. The cluster analysis and ML phylogenetic tree
showed a geographical pattern for E. tetralix supporting a post-glacial migration from Iberia to Ireland. In contrast,
Irish populations of E. mackayana were supported in independent clades in the phylogenetic tree and shared
clusters with Iberian populations in the structure analysis, and FST values were lower among Irish and Spanish
populations than among Irish ones. This suggests that Irish populations of E. mackayana are the result of recent
multiple independent introductions from its native area in northern Spain, probably assisted by humans. However,
the origin of the largest Irish population at Roundstone Bog is unclear and should be further investigated. Post-
glacial, long-distance dispersal is the most plausible explanation for Lusitanian species distribution in Ireland.
ADDITIONAL KEYWORDS: Erica tetralix – Hiberno-Iberian species – Lusitanian species – SNPs.
INTRODUCTION
The current distribution of extant plant species is the
result of past events of migration, colonization and
extinction (Taberlet et al., 1998; Waltari et al., 2007).
Climatic changes, such as the Pleistocene glaciations
in Europe, have forced geographical range contraction
and local extinction of species in northern areas,
followed by northward expansions from southern
refugia after the retreat of the ice (Taberlet et al.,
1998; Hewitt, 2000). On islands, these past cold
events have caused massive local extinctions and
left bare emerged land to be colonized from adjacent
populations (Bennike, 1999; Fernández-Palacios et al.,
2016).
The post-glacial migration history of many plants
and animals across Eurasia is still unknown. An
example is the disjunct distribution of the Lusitanian
(also known as Hiberno-Iberian) elements, a recognized
remarkable phenomenon of European biogeography.
These floristic and faunistic elements occur in the west
of Ireland and the south of Atlantic Europe, mainly
in the north of the Iberian Peninsula, but nowhere
else in Britain and Ireland (Perring, 1967; Webb,
1955; Moore, 1987; Preston & Hill, 1999). Why these
species form isolated populations and, specifically, the
*Corresponding author. E-mail: jaime.fagundez@udc.es
© 2023 The Linnean Society of London.
This is an Open Access article distributed under the terms of the Creative Commons Attribution-
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2 J. FAGÚNDEZ & P. DÍAZ-TAPIA
© 2022 The Linnean Society of London, Botanical Journal of the Linnean Society, 2022, XX, 1–12
origin of the extant Irish populations, has long been a
subject of debate (Corbet, 1962; Moore, 1987). Three
main hypotheses have been put forward that can be
summarized as (1) the existence of a glacial refugium
in Ireland, including land submerged today, or post-
glacial colonization, either (2) through a terrestrial
route, or (3) a long-distance dispersal event (Beatty &
Provan, 2013).
The survival of viable populations in Ireland during
the last glaciation has been historically advocated by
many authors, for all or at least some of the Lusitanian
species (Stapf, 1911; Praeger, 1932; Webb, 1955).
However, during the Pleistocene ice ages the island was
completely covered by an ice sheet, so that the potential
habitats for in situ survival of terrestrial plants and
animals were extremely restricted, particularly during
the Last Glacial Maximum (LGM; 21 000–18 000 BP;
Westley & Edwards, 2017; Clark et al., 2018; Roberts et
al., 2020), when mean temperatures were 7.0±1.0 ºC
lower than the pre-industrial last millennium average
(Osman et al., 2021).
Alternatively, other authors have suggested that
the origin of the Lusitanian elements in Ireland could
be the result of a post-glacial migration (Reid, 1899,
1911; Corbet, 1962). This could have happened as
a long-distance dispersal event or via land bridges.
A terrestrial migration of these species to western
Ireland would have been followed by a local extinction
in Britain and eastern Ireland in more recent times.
Land bridges between Britain and Ireland did not
last beyond the retreat of the ice (Edwards & Brooks,
2008), and thus migration would have happened
under extremely cold conditions. These land bridges
allowed the colonization of Britain and Ireland by
widespread northern species, such as Quercus robur
L. (Kelleher et al., 2004), but a similar process is less
likely for southern species with limited tolerance to
low temperatures.
An alternative migration route would have been
from Iberia by long-distance dispersal, most probably
mediated by humans, but also potentially by birds that
could have transported seeds (Bennike, 1999; Popp et
al., 2011). The hypothesis of a recent long-distance
dispersal event has been put forward by several authors,
but long criticized and dismissed as inconceivable
(Reid, 1899; Praeger, 1934; Webb, 1955). Other
studies have proposed that modern human-mediated
introductions, either inadvertent or deliberate, are
responsible for Ireland’s colonization by flora and
fauna. For example, numerous exotic species have
been introduced and naturalized in modern times [e.g.
Rhododendron ponticum L. (Milne & Abbott, 2000)].
Others were introduced much earlier. For example,
the low haplotypic diversity of Irish populations of the
snail Cepaea nemoralis and the mitochondrial DNA
lineage shared with Pyrenean populations support an
ancient introduction by humans (Grindon & Davison,
2013). Divergence time could be as old as 8000 years
for this snail, but this is consistent with dating of
human colonization of Ireland after the LGM, at least
12 000 BP (Dowd & Carden, 2016).
The most recent studies performed on several
Lusitanian taxa using molecular techniques have
consistently suggested a scenario of post-glacial
dispersal events to Ireland for a range of species
(Grindon & Davison, 2013; Beatty et al., 2015; Reich et
al., 2015; Santiso et al., 2016a, b). Beatty and colleagues
analysed chloroplast and nuclear molecular markers
coupled with climatic niche modelling to study some
of the most remarkable Lusitanian plants, including
Saxifraga spathularis Brot., Pinguicula grandiflora
Lam., Daboecia cantabrica (Huds.) K.Koch and
Euphorbia hyberna L. (Beatty & Provan, 2013, 2014;
Beatty et al., 2015). Low levels of genetic diversity,
the absence of private haplotypes in Irish populations
and their affinity with continental populations did
not support, with different levels of uncertainty, the
hypothesis of the existence of a glacial refugium in
Ireland.
Recent advances in genomics have facilitated the
analysis of large single nucleotide polymorphism (SNPs)
datasets for non-model organisms. Compared with
previously used molecular markers, the application of
these genomic tools has provided greater resolution for
differentiating populations and for the analysis of the
genetic structure in a wide variety of taxa (Andrews
et al., 2016; Parchman et al., 2018). Likewise, these
tools may help to clarify the phylogeography of the
Lusitanian flora, as recommended for fauna (Carlsson
et al., 2014).
Erica mackayana Bab. (Ericaceae) is one of the
most remarkable elements of the Lusitanian flora.
It inhabits two disjunct areas: northern Spain
and western Ireland. In Spain, it is frequent and
even dominant in wet heathlands and bogs on the
Cantabrian and northern Atlantic coasts and in
mountain ranges of up to 1000 m a.s.l. (Fagúndez, 2006,
2016). In western Ireland, it grows on a few wet heaths
and peat bogs of the coasts of Kerry, Mayo, Galway
and Donegal (Sheehy Skeffington & Van Doorslaer,
2015; Sheehy Skeffington, 2017). Fossil remains of E.
mackayana have been recovered in Ireland from the
Gortian interglacial period (Jessen et al., 1959) and
the Late Quaternary period (Jessen, 1949). The species
is sexually sterile in Ireland, only propagating itself
vegetatively (Nelson, 2011).
Erica tetralix L., a close relative of E. mackayana,
has a markedly different distribution, as it is one of
the most widespread European heathers occurring in
Western Europe from the southern Iberian Peninsula
to Scandinavia, and it is frequent throughout Britain
and Ireland (Nelson, 2011). These two species and Erica
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PHYLOGEOGRAPHY OF ERICA MACKAYANA 3
© 2022 The Linnean Society of London, Botanical Journal of the Linnean Society, 2022, XX, 1–12
andevalensis Cabezudo & J.Rivera, a narrow endemic
from the south of the Iberian Peninsula, form a well-
supported clade in the genus (Mugrabi de Kuppler et
al., 2015). In addition, the hybrid between E. tetralix
and E. mackayana [Erica × stuartii (Macfarl.) Mast.]
is frequent in the Irish populations and occasionally
occurs in some overlapping populations in northern
Spain (Fagúndez, 2006; Sheehy Skeffington, 2015,
2017). These species are diploids with a chromosome
number of 2n = 24, and polyploidy has not been
reported in European species of Erica L. (Mugrabi de
Kuppler et al., 2015).
In this study we aimed to clarify the origin of the
isolated Irish populations of E. mackayana. We applied
a genome-wide approach using SNPs to analyse the
phylogeny, genetic structure and genetic diversity
of Iberian and Irish populations. We also included
its close relative E. tetralix in our study, to compare
patterns in species with contrasting present-day
geographical distributions. The origin of the Irish
populations of E. mackayana could be either a case of
in situ survival during the last ice age, the result of
independent multiple long-distance dispersal events
from the northern Iberian Peninsula or the gradual
fragmentation of a single large population settled from
the same region after the LGM. The sexual sterility
of this species and the high rates of hybridization
with E. tetralix in Ireland could be a sign of a strong
bottleneck and could constrain its ability to propagate,
supporting the recent introduction hypothesis (Sheehy
Skeffington, 2017). Moreover, molecular analyses by
Kingston & Waldren (2006) and Pene Eftonga (2013)
were inconclusive but congruent with a founder effect
of the different Irish populations. Another hypothesis
would be a coastal migration route established through
France and southern England, including submerged
land, followed by extinction in the connecting area.
Such a hypothesis has been proposed for explaining
the distribution of other Lusitanian species [e.g. D.
cantabrica (Beatty & Provan, 2013)] based on historical
climatic niche reconstruction. Here we evaluate these
hypotheses and the native status of E. mackayana in
Ireland, which has recently been questioned in favour
of modern human-mediated introductions, based on
historical evidence of trading between both countries
(Sheehy Skeffington, 2015, 2017).
MATERIAL AND METHODS
Studied material
Sixty-five and 53 samples of E. mackayana and E.
tetralix, respectively, were collected in the north of
Spain and the west of Ireland between 2013 and 2017
(Table 1). We sampled all the known populations of E.
mackayana in Ireland (Sheehy Skeffington, 2017) and
six populations in the north of Spain. Erica tetralix
was collected when it co-occurred with E. mackayana.
Additionally, one sample of Erica ciliaris L., the sister
species of the E. tetralix-E. mackayana clade (Mugrabi
de Kuppler et al., 2015), was collected to be used as
an outgroup in the phylogenetic analysis. A flowering
branch of each plant was dried in a bag with silica gel
for DNA extraction, and the main part was mounted
as a herbarium specimen to be stored in the herbarium
of the University of Santiago de Compostela (SANT).
Genomic DNA was extracted from the silica-
preserved samples (Table 1) using an adapted
cetyltrimethylammonium bromide (CTAB) protocol
(Doyle & Doyle, 1987). Genomic DNA was used
to construct nextRAD genotyping-by-sequencing
libraries (SNPsaurus LLC, Eugene, OR, USA)
following Russello et al. (2015). Genomic DNA was
first fragmented with Nextera reagent (Illumina
Table 1. Collection site information and number of samples of the two studied species included in the final analyses
(excluding five specimens with high levels of missing data). Galway includes samples from Carna (numbers in
parentheses). One sample of E. ciliaris, which served as an outgroup for the ML phylogenetic analysis, was collected from
the Pimiango population. NP = not present.
Population Region/ country Latitude (°N) Longitude (°W) Elevation (m a.s.l.) E. mackayana (N) E. tetralix (N)
Pimiango Asturias 43.39 4.54 122 2 NP
Peñas Asturias 43.65 5.85 98 2 NP
Espina Asturias 43.39 6.31 688 13 1
Bustantigo Asturias 43.33 6.71 1011 7 11
Xistral Galicia 43.43 7.48 753 8 NP
Loba Galicia 43.28 7.94 632 10 13
Donegal Ireland 55.03 8.16 57 3 10
Mayo Ireland 54.11 9.52 85 3 4
Galway Ireland 53.43 9.89 22 9(+1) 7(+2)
Kerry Ireland 51.93 10.07 120 3 4
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4 J. FAGÚNDEZ & P. DÍAZ-TAPIA
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Inc, San Diego, CA, USA), which also ligates short
adapter sequences to the ends of the fragments. The
Nextera reaction was scaled for fragmenting 15ng of
genomic DNA. Fragmented DNA was then amplified
for 27 cycles at 74 °C, with one of the primers
matching the adapter and extending nine nucleotides
into the genomic DNA with the selective sequence
GTGTAGAGCC. Thus, only fragments starting with
a sequence that can be hybridized by the selective
sequence of the primer will be efficiently amplified.
The nextRAD libraries were sequenced on an Illumina
HiSeq 4000 System with two lanes of 150-bp reads (at
the University of Oregon).
The genotyping analysis used custom scripts
(SNPsaurus LLC) that trimmed the reads using
bbduk (BBMap tools v.38.79, http://sourceforge.net/
projects/bbmap/) with the following parameters: ktrim
= r, k = 17, hdist = 1, mink = 8, minlen = 100, ow = t,
qtrim = r, trimq = 10. Next, a de novo reference was
created by collecting 10 000 000 reads in total, evenly
from the samples, and excluding reads that had fewer
than six or more than 700 counts. The remaining loci
were then aligned to each other using bbmap (BBMap
tools) to identify allelic loci and collapse allelic
haplotypes to a single representative. All reads were
mapped to the reference using bbmap (BBMap tools)
with the following parameters: minid = 0.95, ambig
= toss and maxindel = 8. Genotype calling was done
using callvariants (BBMap tools) with the following
parameters: ploidy = 2, multisample = t, rarity = 0.05,
minallelefraction = 0.05, usebias = f, ow = t, nopassdot
= f, minedistmax = 5, minedist = 5, minavgmapq = 15,
minreadmapq = 15, minstrandratio = 0.0, strandedcov
= t. The mean number of reads was 4 193 195.69
(SD 2477 783.97), and the mean of retained reads
was 1 597 540.16 (SD 969 414.05) after filtering and
mapping. The variant call format (vcf) was filtered to
remove alleles with a population frequency of > 3%.
We obtained an aligned matrix of 2767 polymorphic
loci. Five samples with more than 50% of missing data
were excluded from further analyses, so that the final
dataset comprised 52 and 61 samples of E. tetralix and
E. mackayana, respectively.
Phylogenetic analySiS
The vcf file was exported as a fasta file using Tassel,
and the heterozygous sites were encoded following the
International Union of Pure and Applied Chemistry
(IUPAC). Invariant sites were filtered using IQtree
v.2.1.3 so that the final alignment included 2051
base pairs. We built a ML phylogenetic tree using
RAxML-NG v.8.2.12 (Kozlov et al., 2019), using a
GTGTR4 substitution model, a G among-site rate
heterogeneity model and the ascertainment bias
correction ASC_LEWIS, with 1000 non-parametric
bootstrap replicates.
analySiS of genetic diverSity and PoPulation
genetic Structure
VCFtool S v. 0.1.13 (Danecek et al., 2011) was used
to prune out linked loci using 150bp as the minimum
distance between two sites, so that the final dataset
comprised 791 and 747 variant sites for E. mackayana
and E. tetralix, respectively. The overall level of
missingness across all sites was 16.63% and 15.96%
in the E. mackayana and E. tetralix data sets,
respectively. Average expected (Hs) and observed (Ho)
heterozygosity, and inbreeding coefficient (FIS) were
estimated using hierfstat v.0.04-6 (Goudet, 2005). Weir
and Cockerham pairwise FST values among populations
and their statistical significance were calculated using
the R package SambaR v.1.06 with 1000 bootstrap
replications (de Jong et al., 2021).
The population genetic structure was separately
analysed for the two studied species using two
complimentary approaches. Firstly, a ML population
structure assessment was inferred using ADMIXTURE
v.1.3.0 with default parameters (Alexander & Lange,
2011) to estimate individual ancestries (Q matrix)
for potential populations (K) ranging from K = 1 to 8
and 11 for E. mackayana and E. tetralix, respectively.
Twenty independent analyses were run. The online
web server of the program CLUMPAK (Kopelman et
al., 2015) was used to summarize the ADMIXTURE
results into structure plots. Secondly, we analysed
patterns of geographical differentiation using a
principal component analysis (PCA) conducted with
the R package adegenet v.2.1.3 (Jombart, 2008).
RESULTS
Phylogenetic analySeS
In the ML phylogenetic tree, E. mackayana and E.
tetralix were retrieved in two supported clades (Fig. 1).
For the widespread species E. tetralix the tree shows
a clear geographical pattern, as samples from Ireland
were resolved in a neatly defined clade that received
full support, whereas Spanish samples were placed in
several earlier diverged lineages.
The Asturian populations (Bustantigo and Espina)
were clustered in a clade nested in the basal Galician
(Loba) population, and sister to the Irish clade. The
topology of the Irish clade was structured according to
the geographical distribution of populations following
a latitudinal pattern, from Kerry in the south to
Donegal in the north, with different levels of support
for each population (Fig. 1).
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PHYLOGEOGRAPHY OF ERICA MACKAYANA 5
© 2022 The Linnean Society of London, Botanical Journal of the Linnean Society, 2022, XX, 1–12
Ericatetralix
Ericamackayana
Kerry
Mayo
Galicia
(Loba)
Mayo
Carna
Galway
+ Carna
Kerry
88
Espina
Ericaciliaris (outgroup)
Asturias
(Bustantigo)
Donegal
Galway
Xistral
Donegal
Xistral
Loba
Asturias
(Peñas +
Bustantigo)
Galicia
(Xistral +
Loba)
100
67
50
Bootstrap nodevalues
Asturias
(Pimiango+
Espina +
Bustantigo)
E
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Figure 1. RAxML phylogenetic tree of the studied populations of E. tetralix and E. mackayana, with colours and labels
for each population/region as in the maps. Codes are for samples, bootstrap node values are proportional to circle sizes (see
legend).
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6 J. FAGÚNDEZ & P. DÍAZ-TAPIA
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The tree for E. mackayana was opposite to that of
E. tetralix for the Iberian populations, with Galician
populations (Xistral and Loba) nested in the early-
diverging clade of Asturias (Pimiango, Peñas, Bustantigo
and Espina). The four Irish populations, Roundstone
Bog (Galway), Donegal, Kerry and Mayo, were placed in
four strongly supported clades (node bootstrap value =
100), intermixed with the Iberian populations and not
related to each other (Fig. 1). One sample collected in
Carna, initially assigned to the Galway population as it
was collected close to the Roundstone Bog population,
was also placed independently in the tree.
genetic diverSity and PoPulation genetic
Structure
Genetic diversity was in general higher in Spanish
populations compared with Irish ones in the two
studied species (Table 2). The inbreeding coefficient
(FIS) was negative for the two species in all sampling
sites, indicating a small excess of heterozygotes.
The FST estimates showed relevant differences in
patterns of genetic differentiation of populations
between species. Differentiation among populations
within each of the studied countries, Spain and Ireland,
was lower (≤ 0.16) than between populations of these
two countries (≥ 0.3) in E. tetralix (Table 3). Similarly
low FST estimates were found among populations of
E. mackayana from Spain (≤ 0.12, Table 4). However,
FST estimates among Irish populations were similar
or higher (≥ 0.36) when compared with Spanish
populations (0.11–0.35). In particular, two Irish
populations (Mayo and Kerry) had low FST estimates
(0.11–0.31) with regard to Spanish populations. In
contrast, some Irish populations showed the highest
values (e.g. Kerry vs. Donegal, 0.43).
The clustering analysis revealed a highly structured
genetic assemblage in populations for the two species
(Fig. 2). According to the cross validations (Supporting
Information, Fig. S1), the strongest support was
found for two and three clusters in E. mackayana
and E. tetralix, respectively, but cross validation
values were highly similar in both species for K = 2–3
(ranging between 0.331–0.332 and 0.369–0.39 in E.
mackayana and E. tetralix, respectively). However, the
correspondence between clusters and the geographical
origin of samples differed greatly between species.
In E. tetralix, the three clusters corresponded to the
three main geographical areas [Ireland (blue), Galicia
(green) and Asturias (red)] with limited admixture
(Fig. 2A, C, E). The single plant analysed from Espina,
in Asturias, showed an admixture of Galician and
Asturias clusters. Irish populations had an increasing
admixture of the Spanish clusters from north to south.
In E. mackayana, the three clusters identified did
not correspond with the geographical origin of samples
(Fig. 2B, D, F). One of the clusters (red) was dominant
in plants from Galicia (Loba and Xistral populations)
and the Irish populations from Kerry, Carna and
Donegal. A second cluster (green) included plants from
Asturias (Bustantigo, Espina, Peñas and Pimiango)
Table 2. Estimates of genetic diversity calculated across sampling locations in which more than one sample was collected
E. tetralix E. mackayana
Population N Ho Hs FIS N Ho Hs FIS
Donegal 10 0.205 0.169 -0.075 3 0.139 0.135 -0.045
Mayo 4 0.124 0.174 -0.09 3 0.222 0.153 -0.114
Carna 2 0.192 0.168 -0.055 1 - - -
Galway 7 0.204 0.171 -0.072 9 0.26 0.176 -0.167
Kerry 4 0.243 0.179 -0.125 3 0.232 0.159 -0.123
Loba 13 0.253 0.208 -0.089 10 0.29 0.235 -0.114
Xistral - - - - 8 0.282 0.239 -0.093
Bustantigo 12 0.265 0.217 -0.099 7 0.29 0.244 -0.093
Espina 1 - - - 13 0.276 0.227 -0.098
Peñas - - - - 2 0.189 0.188 -0.024
Pimiango - - - - 2 0.3 0.229 -0.149
Table 3. Pairwise FST estimates between E. tetralix
populations, with 1000 bootstrap replicates. The colour
gradient from green to red indicates increasing FST values.
FST P-values were 0 for all pairwise estimates
Galway Kerry Mayo Donegal Loba Bustantigo
Kerry 0.07
Mayo 0.06 0.10
Donegal 0.09 0.14 0.06
Loba 0.35 0.30 0.32 0.36
Bustantigo 0.34 0.30 0.31 0.34 0.16
Espina 0.37 0.35 0.36 0.38 0.12 0.05
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PHYLOGEOGRAPHY OF ERICA MACKAYANA 7
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and the Irish population from Mayo. Some admixture
of these two groups was detected in plants from these
sites. The third cluster (blue) was represented by
plants from Galway, and was marginally represented
in the Galician populations of Xistral and Loba, the
Asturian population of Espina and the Irish population
of Donegal.
We further evaluated population structure using a
PCA. Consistent with the structure clustering analysis,
patterns again clearly differed between the two species.
In E. tetralix, PC1 clearly differentiated Spanish and
Irish populations, whereas PC2 and PC3 separated
populations in each of these two regions (Fig. 3A). PC2
differentiated two groups of samples, corresponding to
Galician (Loba) and Asturian samples. Likewise, PC3
moderately distinguished four groups of Irish samples,
the placement of which in the PCA is congruent with
their latitudinal origin. In contrast, in E. mackayana
PC1 clearly separated the Galway (Roundstone
Bog) population from all other Irish and Spanish
samples (Fig. 3B). Moreover, PC2 and PC3 moderately
distinguished north-western and northern Spanish
populations, whereas the remaining Irish populations
overlapped or were placed close to Spanish ones; the
population from Kerry was the only exception.
DISCUSSION
Our study revealed conspicuous differences in
population genetic structure between the widespread
Atlantic E. tetralix and its restricted congener E.
mackayana. We found a strong phylogeographic signal
in populations of E. tetralix, congruent with a south-
north migration and isolation-by-distance pattern. In
contrast, the population structure of E. mackayana
did not correspond with the geographical distribution
of samples, and our analyses suggest that most Irish
populations are the result of multiple recent dispersal
events from different Spanish populations.
In E. tetralix, Irish and Iberian populations
were clearly differentiated in all the analyses. The
southernmost population in Ireland (Kerry) included a
small percentage of the two Iberian clusters which were
gradually lost northwards. Also, the genetic diversity
was higher in Iberian populations. This pattern is
consistent with the classical post-glacial migration from
southern refugia to northern Europe described for other
species (Hewitt, 2000; Kelleher et al., 2004), including
the most widespread heather Calluna vulgaris (L.) Hull.
This species showed a genetic diversity decrease and a
strong isolation-by-distance signal from south to north,
from Iberia to northern Europe and at a regional scale in
Scotland (Mahy et al., 1999; Rendell & Ennos, 2002; Gil-
López et al., 2022). Although our study included only a
limited number of populations of E. tetralix, considering
its wide geographical range, the pattern found in the
phylogenetic analysis is consistent with a south-north
migration both at the broad scale (Iberia to Ireland)
and the regional scale (Irish populations). However,
additional populations covering the entire distribution
of E. tetralix will be required to further understand the
phylogeography of this widespread Atlantic species in a
wider geographical context.
In contrast to E. tetralix, Spanish and Irish
populations of E. mackayana were not discriminated
at the country level; however, the Roundstone Bog
(Galway) population was clearly distinct from the
others. The phylogenetic tree showed a basal grade of
Asturian populations with Galician ones nested in it
with low support. The Irish populations were placed
in unrelated positions among them. Surprisingly, the
single specimen collected in Carna was genetically
more similar to other populations albeit being only c. 15
km apart from the large Roundstone Bog population in
Galway. Instead, Carna was placed independently from
other Irish populations in the phylogenetic tree, and
shared a cluster with Kerry, Donegal and populations
from Galicia (Xistral, Loba) in the structure analysis.
Table 4. Pairwise FST estimates between E. mackayana populations, with 1000 bootstrap replicates. The colour gradient
from green to red indicates increasing FST values. One plant from Carna was not included. FST P-values were 0.01 for
Bustantigo and Pimiango, 0 for all other pairwise estimates
Donegal Galway Kerry Mayo Loba Xistral Bustantigo Pimiango Espina
Galway 0.42
Kerry 0.43 0.36
Mayo 0.4 0.38 0.37
Loba 0.2 0.22 0.16 0.15
Xistral 0.2 0.21 0.16 0.16 0.02
Bustantigo 0.19 0.24 0.16 0.11 0.04 0.04
Pimiango 0.29 0.3 0.28 0.27 0.06 0.05 0.02
Espina 0.21 0.23 0.19 0.13 0.06 0.06 0.03 0.04
Peñas 0.34 0.35 0.31 0.26 0.09 0.1 0.06 0.12 0.07
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8 J. FAGÚNDEZ & P. DÍAZ-TAPIA
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DMGCKL B
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Donegal
Mayo
Galway
Carna
Kerry
Pimiango
Peñas
Espina
Bustango
Loba
Xistral
Espina
Bustango
Loba
Figure 2. Population structure genetic matrix and geographical distribution of each cluster for populations of E. tetralix (A,
C, E) and E. mackayana (B, D, F). K-values were selected based on cross-validation: E. mackayana and E. tetralix K = 3. Size of
the circles in the maps are proportionate to number of samples per population. Y axes in E and F represent frequency (0-1). D =
Donegal, M = Mayo, G = Galway, C = Carna, K = Kerry, L = Loba, X = Xistral, B = Bustantigo, E = Espina, P = Peñas, I = Pimiango.
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PHYLOGEOGRAPHY OF ERICA MACKAYANA 9
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Finally, the population of Mayo shared a cluster with
the Asturian populations in the structure analysis,
and it was placed among the Asturian samples in the
phylogenetic tree, sharing an unsupported clade with
plants from Peñas, in coastal central Asturias.
Our findings support the recent dispersal hypothesis
for most Irish E. mackayana populations from multiple
independent events. Trading of goods in the 19th and
20th centuries could have been a potential path of
human-mediated introduction (Sheehy Skeffington,
2015; Sheehy Skeffington & Van Doorslaer, 2015).
Smugglers at that time did not use the main roads,
and they may have used heather to protect cautiously
hidden goods in isolated places away from the
main paths and at some distance from the coast,
corresponding to the present-day location of most Irish
E. mackayana populations (Sheehy Skeffington, 2017).
Other references to the use of heathers in trade
would support this hypothesis. For example,
Reich et al. (2015) claimed that the slug Geomalacus
maculosus, another Lusitanian species, may have
been introduced unintentionally in materials used
for packaging wine, which ‘…were probably protected
by layers of heath or other vegetation which may
have contained G. maculosus specimens and/or
eggs’. Similarly, Foss & Doyle (1988) suggested that
pilgrims in the 15th century may have introduced
Erica erigena R.Ross in Ireland from Galicia, another
heather species with a Lusitanian distribution. In
another study, Santiso et al. (2016a, b) included an
Irish population of Arbutus unedo L., one of the most
iconic examples of the Lusitanian flora, in their wide
phylogeographic study of the species using non-
coding plastid DNA and amplified fragment length
polymorphisms (AFLPs). The close linkage of Irish to
northern Iberian populations is coherent with a long-
distance colonization event, rather than a terrestrial
colonization or in situ survival during the LGM as
supported by previous authors (Sealy & Webb, 1950;
Webb, 1955). An alternative hypothesis of introduction
through mining activities has been given by Sheehy
Skeffington & Scott (2021).
An alternative explanation for the foundation
of Irish E. mackayana populations in the absence
of human intervention could be a long-distance
dispersal probably facilitated by migrant birds, as
suggested for C. vulgaris (Mahy et al., 1999). The
seeds of C. vulgaris and many Erica spp., including E.
mackayana, are small (< 1mm), light (< 0.05mg), have
a prominent ornamentation and are produced in large
numbers (Fagúndez et al., 2010). These traits may
facilitate long-distance travelling trapped among bird
feathers, although they lack any specific structure for
epizoochory (Sorensen, 1986).
Irish populations of E. mackayana, except
Roundstone Bog (Galway), were genetically similar
to Spanish plants from different regions suggesting
that plants from Mayo could have recently dispersed
from the coast of Asturias, whereas plants from
Donegal, Carna and Kerry could have come from
Galicia. This is in agreement with previous studies
based on an AFLP analysis that revealed a mixed
cluster of Irish and Spanish populations, interpreted
as a potential effect of multiple founder events
(Kingston & Waldren, 2006). However, the authors
stated that samples could have been misidentified,
and plants from Donegal could be of hybrid origin
(Kingston & Waldren, 2006). Similarly, results from
Pene Eftonga (2013) provided consistent results with
the different origins of Irish populations, which also
showed low genetic diversity compared to those from
Spain.
The genetic differentiation among Irish populations,
revealed by the highest FST values observed in the
species, suggests that they are isolated. This result
Bustantigo
C
ar
n
a
D
one
g
a
l
E
s
pi
na
G
alwa
y
K
err
K
K
y
Loba
Ma
y
o
P
e
ñ
as
Pi
m
i
an
g
o
Xis
tr
al
PC1 (12.1%)
PC2 (4.9%)
PC1 (30%)
PC2 (7.8%)
PC1 (30%)
PC3 (3.9%)
PC1 (12.1%)
PC3 (4.2%)
0
30
10
20
PCA eigenvalues
PCA eigenvalues
0
30
10
20
0
80
40
IRELAND N SPAIN N SP N SP NW SPAIN NW SP NW SP
Erica tetralix
Erica mackayana
A
B
Figure 3. Principal component analysis (PCA) showing
individual variation of the genomic data. Panels show
PC1 plotted against PC2 and PC3 for E. tetralix (A) and
E. mackayana (B). The amounts of variation explained by
each PC are given as percentages. Eigenvalues for the first
ten PCs obtained are shown in the insets.
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10 J. FAGÚNDEZ & P. DÍAZ-TAPIA
© 2022 The Linnean Society of London, Botanical Journal of the Linnean Society, 2022, XX, 1–12
is unsurprising considering that the species does not
set viable seeds in Ireland (Webb, 1955; Nelson, 2011),
contrary to Iberian populations (Fagúndez & Izco,
2010). In fact, Irish populations have been described
as single clones, and sexual reproduction only takes
place through crossing with E. tetralix (Nelson, 2005;
Sheehy Skeffington & Van Doorslaer, 2015). The
hybrid is frequent in Ireland but not in Spain where
the contact area between the species is narrow, and
even in co-occurring populations the hybrid is rare
(Fagúndez, 2006). This markedly different pattern in
hybridization rates between countries resembles other
introduced plant species that frequently hybridize
in non-native areas but rarely in their home range
(Ellstrand & Schierenbeck, 2000; Prunera-Olivé et al.,
2019).
Our results showed a conspicuous differentiation
of the population from Roundstone Bog (Galway)
compared with the other Irish and Iberian populations.
However, the phylogenetic tree showed this population
nested in the main clade similar to the other Irish
populations, and not as a sister clade, suggesting
a similar origin to the others. The glacial refugium
hypothesis is not supported by these results. There
are fossil deposits of E. mackayana in Ireland, dating
from both the Interglacial Gortian period (365 000
BP) and post-glacial times (Jessen, 1949; Jessen et al.,
1959). However, other non-native species registered in
the fossil record are known to have been introduced
recently in Ireland, such as R. ponticum (Jessen, 1948;
Milne & Abbott, 2000). Several species, including E.
mackayana and other heathers such as Erica scoparia
L. and Erica umbellata L., are known to have occurred
at northern latitudes during pre- and interglacial
times, but did not recover their former range after the
last glaciation (Jessen et al., 1959, Wilson et al., 1973).
Sterility and hybridization are also major constraints
for long-term population survival, even if vegetative
and clonal growth is intense (Wolf et al., 2001). The
current Roundstone Bog (Galway) population could
be another recent introduction from the Iberian
Peninsula, but the source may have not been captured
in our sampling, explaining the strong differences of
this population shown in the structure analysis and
the PCA. We covered the species range in the Iberian
Peninsula, but the species inhabits several mountain
ranges with some potential degree of isolation
(Fagúndez, 2006, 2016).
CONCLUSION
Our results demonstrate, with a high level of
confidence, that Irish populations of E. mackayana
have independent origins and are not the result of
fragmentation of a larger population. This confirms it
is not a relict species that expanded its distribution
from an Irish glacial refugium. However, there is
some uncertainty about the origin of the largest Irish
population at Roundstone Bog (Galway), which should
be investigated further by dense sampling in northern
Spain. The high resolution of the SNPs showed that
Irish populations are probably the result of multiple
independent colonization events, supporting the
hypothesis of long-distance dispersal events for the
Lusitanian flora and fauna, in line with recent works
that found similar patterns for other elements with
disjunct distributions (Grindon & Davison, 2013; Reich
et al., 2015; Beatty et al., 2015). We suggest this is
probably the biogeographic pattern for the majority or
all of the Lusitanian species. Dating the arrival of these
species and disentangling how humans may have been
responsible of their introductions in historical times is
another challenge for Iberian and Irish biogeography.
ACKNOWLEDGEMENTS
We would like to thank M. Sheehy Skeffington and R.
Sheppard who provided plant samples for this study,
Alexander Papadopulos who commented on an early
draft of the paper and Rodolfo Barreiro who helped
us with the SNPs analyses; we also acknowledge the
computational facilities of Centro de Supercomputación
de Galicia (CESGA).
funding
This work was partially supported by Xunta de Galicia
‘Talento Senior’ (grant 03IN858A2019-1630129) to
P.D-.T. and ‘Axudas para a consolidación e estruturación
de unidades de investigación competitivas do SUG’
(grants ED431D 2017/20, ED431B 2018/49). Funding
for open access charge: Universidade da Coruña/
CISUG.
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SUPPORTING INFORMATION
Additional supporting information may be found in the online version of this article on the publisher’s website.
Figure S1. ADMIXTURE mean cross validation values across twenty independent runs for K = 1–8 and 1–11 in
E. tetralix and E. mackayana, respectively.
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