Ci a ion: Suá ez-San iago, V.N.;
P o an, J.; Rome o-Ga cía, A.T.;
Ben-Menni Schule , S. Gene ic
Di e si y and Phylogeog aphy o he
Relic T ee Fe n Culci a mac oca pa:
In luence o Clonali y and B eeding
Sys em on Gene ic Va ia ion. Plan s
2024,13, 1587. h ps://doi.o g/
10.3390/plan s13121587
Academic Edi o s: Jacek U baniak
and Paweł Kwia kowski
Recei ed: 12 Ap il 2024
Re ised: 2 June 2024
Accep ed: 5 June 2024
Published: 7 June 2024
Copy igh : © 2024 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license (h ps://
c ea i ecommons.o g/licenses/by/
4.0/).
plan s
A icle
Gene ic Di e si y and Phylogeog aphy o he Relic T ee Fe n
Culci a mac oca pa: In luence o Clonali y and B eeding Sys em
on Gene ic Va ia ion
Víc o N. Suá ez-San iago 1,* , Jim P o an 2,†, Ana Te esa Rome o-Ga cía1and Sami a Ben-Menni Schule 1,*
1Depa men o Bo any, Facul y o Sciences, Uni e si y o G anada, 18071 G anada, Spain; a ome o@ug .es
2Ins i u e o Biological, En i onmen al and Ru al Sciences, Abe ys wy h Uni e si y,
Abe ys wy h SY23 3DA, UK
*Co espondence: sua ez@ug .es (V.N.S.-S.); sami a@ug .es (S.B.-M.S.)
†In memo iam J. P o an, es in peace.
Abs ac : The ee e n Culci a mac oca pa, a h ea ened Ibe ian–Maca onesian endemism, ep e-
sen s he sole Eu opean species o he o de Cya heales. Conside ed a Te ia y elic o Eu opean
Palaeo opical lo a, i s e olu iona y his o y and gene ic di e si y, po en ially in luenced by p e-
sumed high clonal p opaga ion, emain la gely unknown. This s udy elucida es he phylogeog aphic
his o y o C. mac oca pa, assessing he impac o ege a i e ep oduc ion on popula ion dynamics and
gene ic a iabili y. We p o ide gene ic da a om eigh newly iden i ied nuclea mic osa elli e loci
and one plas id DNA egion o 17 popula ions spanning he species’ ange, oge he wi h species
dis ibu ion modeling da a. Mic osa elli es e eal pe asi e clonali y in C. mac oca pa, which has
a ied among popula ions. We assess he impac o clonali y on gene ic di e si y and e alua e how
es ima es o in a-popula ion gene ic di e si y indices and gene ic s uc u ing a e a ec ed by he
chosen de ini ion o “indi idual” ( ocusing exclusi ely on gene ically dis inc indi iduals, gene s, as
opposed o conside ing all independen clonal eplica es, ame s). We iden i y wo main popula ion
g oups, one in he no he n Ibe ian Peninsula and he o he in he Maca onesian a chipelagos and
sou he n Ibe ian Peninsula. Wi hin each g oup, we ound elic popula ions (in he Azo es and he
Can ab ian Co nice) as well as ecen o igina ed popula ions. This popula ion s uc u e sugges s
coloniza ion dynamics in which ecen popula ions o igina ed om one o a ew gene s o elic
popula ions and became es ablished h ough in a-game ophy ic sel - e iliza ion and ege a i e
expansion. DAPC analysis acili a ed he iden i ica ion o alleles ha mos signi ican ly con ibu ed
o he obse ed popula ion s uc u e. The cu en Andalusian popula ions appea o ha e o igina ed
om coloniza ion e en s om he Azo es and he Can ab ian Co nice. Ou indings sugges ha C.
mac oca pa pe sis ed h ough he Las Glacial Maximum in wo e ugia: he Azo es and he Can ab ian
Co nice. Coloniza ion in o new a eas occu ed p esumably om hese e uges, gene a ing wo la ge
popula ion g oups wi h s uc u ed gene ic di e si y. This s udy unde sco es he signi icance o
clonali y in es ablishing new popula ions and shaping gene ic s uc u e.
Keywo ds: b eeding sys em; clonali y; Culci a mac oca pa; e n phylogeog aphy; gene ic di e si y;
glacial e ugia; mic osa elli es; palaeo opical lo a; plas id DNA; species dis ibu ion modeling
1. In oduc ion
Al hough e ns a e he second mos di e se g oup o ascula plan s a e angiospe ms
(ca. 11,000 species; [
1
]), he e is a compa a i e lack o da a on popula ion gene ics and
phylogeog aphy o his impo an g oup [
2
]. Nume ous e ns a e conside ed lineages o
Te ia y o igin, especially hose om he Medi e anean egion [
3
,
4
], which cons i u ed
he main componen o he he baceous laye o he Eu opean lau oid o es [
5
–
7
] and
su i ed he Qua e na y glacia ions in well-cha ac e ized glacial e ugia, such as he
Ibe ian Peninsula, I aly, he Balkans, and G eece [8].
Plan s 2024,13, 1587. h ps://doi.o g/10.3390/plan s13121587 h ps://www.mdpi.com/jou nal/plan s
Plan s 2024,13, 1587 2 o 26
Fe ns, wi h hei high capabili y o spo e-media ed dispe sal [
9
], usually exhibi
s ong popula ion connec i i y and educed gene ic s uc u e [
10
]. Howe e , he limi ed
a ailabili y and disjunc dis ibu ion o sui able habi a s wi h high mois u e and wa m
empe a u es, especially o sou he n Eu opean Te ia y e ns, may esul in p onounced
gene ic s uc u ing (e.g., [
11
–
20
]). In e ns, as in many o he non-seed plan s, e iliza ion is
a pos -dispe sal p ocess (occu ing on he game ophy e om spo es al eady dispe sed), and
consequen ly he p obabili y o colonizing a new habi a as well as he gene ic composi ion
o he new popula ion will depend on he b eeding sys em o he species. The coloniza ion
p ocess will be less p obable o e ns wi h ou c ossing and in e -game ophy ic sel ing,
and he esul ing popula ions will be gene ically mo e di e se. By con as , hose wi h
in a-game ophy ic sel ing will be able o es ablish a new popula ion e en om a single
spo e, bu will esul in popula ions wi h e y low di e si y [
21
,
22
]. Mo eo e , many
e ns also ha e he abili y o p opaga e ege a i ely, u he shaping le els and pa e ns
o wi hin- and be ween-popula ion di e si y. Clonal ep oduc ion will end o dec ease
in wi hin-popula ion gene ic di e si y due o a lack o meiosis and ecombina ion o be-
cause in e clonal compe i ion can lead o he elimina ion o less adap ed clones, e en o
he ex en o o ming monoclonal popula ions [
23
]. Howe e , clonali y ends o inc ease
he e ozygosi y by he accumula ion o mu a ion and di e gence be ween alleles [
24
,
25
].
Popula ion di e en ia ion, es ima ed as he F
ST
alue, is d as ically educed when ep o-
duc ion ends owa d s ic clonali y because gene ic di e si y is main ained wi hin clonal
lineages. Howe e , e en e y limi ed p opo ions o sexual ep oduc ion make he e ec o
clonali y on popula ion di e en ia ion negligible [24].
In he p esen s udy, we ocus on Culci a mac oca pa C. P esl (Culci aceae; Figu e 1), a
diploid ee e n conside ed o be a elic om Palaeo opical lo a [
3
]. I has a suba bo escen -
c eeping habi wi h a hick hizome co e ed wi h eddish ili o m scales. The onds
can exceed 2 m in leng h, and on he ma gins o hese he eni o m so i de elop ([
26
];
Figu e 1). Culci a mac oca pa is an Ibe ian–Maca onesian endemic species (Andalusia, he
Can ab ian Co nice, he Azo es, he Cana y Islands, and Madei a), and he only Eu opean
ep esen a i e o he genus, since he o he known species [C. conii olia (Hook.) Maxon] has
an exclusi ely Ame ican dis ibu ion. Acco ding o o he au ho s [
27
,
28
], he di e gence
be ween he lineages o he wo species occu ed abou 20 million yea s ago. Culci a
mac oca pa is es ic ed o shady si es wi h mild empe a u es h oughou he yea and high
humidi y and soil mois u e, as in alleys nea he coas , usually a al i udes below 300 m,
o in highe a eas associa ed wi h og bel s (e en abo e 1000 m), especially in he Azo es.
As many o hese habi a s whe e C. mac oca pa is dis ibu ed a e unde h ea , and gi en i s
disjunc dis ibu ion, he species is cu en ly p o ec ed unde he Be n Con en ion and he
Habi a s Di ec i e [
29
,
30
]. In addi ion o being an endange ed species in he Red Lis o
Spanish Vascula Flo a, i is also included in he Spanish Lis o Wild Species unde Special
P o ec ion [
31
,
32
]. Also, his species is also conside ed c i ically endange ed in he Red Lis
o he Vascula Flo a o Mainland Po ugal [33].
Rep oduc i ely, cul u e expe imen s ha e shown ha game ophy es o C. mac oca pa
a e ini ially male and la e become he maph odi e, and ha hey do no p oduce an he id-
iogens [
34
,
35
], a scena io ha seems o a o in a-game ophy ic sel ing [
36
,
37
]. A s udy
was pe o med on he gene ic a ia ion in six popula ions om Galicia (no h-wes e n
Ibe ian Peninsula) using 13 enzyma ic sys ems [
38
], seeking o assess he conco dance
be ween geno ypic equencies and he b eeding sys em. Howe e , no insigh s we e gained
in o he ep oduc i e sys em o he species, since only a single mul ilocus geno ype was
ound ac oss all indi iduals and popula ions, al hough e idence was ound o he in ense
clonal ep oduc ion o he species [
38
]. Culci a mac oca pa has a c eeping hizome ha
can exceed one me e in leng h, bi u ca ing and gi ing ise o new shoo apices ha o m
ad en i ious oo s, each shoo apex cons i u ing a sepa a e clone o ame [
35
,
38
,
39
]. The
lack o obse ed gene ic a ia ion was a ibu ed by he au ho s o gene ic d i associa ed
wi h he educ ion in he numbe o popula ions du ing he las ice age, and subsequen
ounde e ec s du ing Holocene expansion [38].
Plan s 2024,13, 1587 3 o 26
Plan s 2024, 13, x FOR PEER REVIEW 3 o 26
he educ ion in he numbe o popula ions du ing he las ice age, and subsequen
ounde effec s du ing Holocene expansion [38].
Figu e 1. Culci a mac oca pa. Indi idual om he Almo aima popula ion in Cádiz p o ince (Anda-
lusia, Spain), exhibi ing he suba bo escen habi . Inse (uppe igh ), de ail o a ond wi h so i.
Pho os: Gab iel Blanca.
The e olu iona y his o y o C. mac oca pa emains unknown. Fu he mo e, as no ed
abo e, molecula gene ic app oaches ha e no as ye been success ully used o e alua e
Figu e 1. Culci a mac oca pa. Indi idual om he Almo aima popula ion in Cádiz p o ince (Andalusia,
Spain), exhibi ing he suba bo escen habi . Inse (uppe igh ), de ail o a ond wi h so i. Pho os:
Gab iel Blanca.
The e olu iona y his o y o C. mac oca pa emains unknown. Fu he mo e, as no ed
abo e, molecula gene ic app oaches ha e no as ye been success ully used o e alua e he
e ec o clonali y on i s in aspeci ic gene ic s uc u e. To quan i y he gene ic a ia ion
and i s dis ibu ion wi hin and be ween popula ions, and o iden i y sui able a eas o
Plan s 2024,13, 1587 4 o 26
he species in he pas and he u u e, we used nuclea mic osa elli e loci, one plas id
ma ke , and species dis ibu ion modeling (SDM) wi h a double aim. Fi s ly, we sough
o in e he species’ phylogeog aphic his o y. The biogeog aphic pa e n o he species,
ypical o a elic axon, makes C. mac oca pa a good sys em o assess he impac o bo h
p e-Pleis ocene and Qua e na y geological and clima ic e en s on popula ion p ocesses
ha gene a e gene ic s uc u e, and p esen insigh s in o how ongoing and u u e clima e
change may shape e olu iona y pa e ns. Fu he mo e, phylogeog aphic da a om ancien
axa, such as C. mac oca pa, may also p o ide insigh s in o he demise o he Palaeo opical
lo a o Eu ope. Secondly, we sough o es ima e he impo ance o sexual and ege a-
i e ep oduc ion on popula ion composi ion and o e alua e he e ec o clonali y on
in aspeci ic gene ic s uc u e.
2. Ma e ials and Me hods
2.1. Plan Ma e ial
A o al o 443 indi iduals o C. mac oca pa was sampled om 17 popula ions in
4 geog aphical egions ac oss i s dis ibu ion ange: Andalusia, he Azo es, he Cana y
Islands, and he Can ab ian Co nice. In Madei a, whe e he species is epo edly also
p esen , we could no ind any popula ion. The numbe o popula ions pe egion was
be ween one and i e, and he numbe o sampled indi iduals pe popula ion a ied om
6 o 30 (Table 1; Figu e 2).
Table 1. Sampling de ails o Culci a mac oca pa popula ions used in he p esen s udy.
Sample Size
Code Loca ion Vouche Geog aphical
Coo dina es
Mic osa elli es
p DNA
Andalusia
ALM Cádiz: Almo aima GDA 65361 N 36.304◦/W 5.520◦6 3
CRM Cádiz: Cabece a del ío de la Miel GDA 65363 N 36.105◦/W 5.528◦30 5
PIN Cádiz: Laja del Pina ejo GDA 65360 N 36.188◦/W 5.589◦30 5
RM Cádiz: Río de la Miel GDA 65359 N 36.112◦/W 5.507◦29 5
SDN Cádiz: Sie a del Niño GDA 65362 N 36.186◦/W 5.610◦30 5
Azo es
CAR Te cei a: Alga do Ca ão GDA 63533 N 38.727◦/W 27.215◦30 5
CID São Miguel: Se e Cidades GDA 63534 N 37.835◦/W 25.788◦29 5
FOG São Miguel: Lagoa do Fogo GDA 63532 30 5
NAT Te cei a: G u a do Na al GDA 63531 N 38.738◦/W 27.264◦30 5
Cana y Isl.
IJU Tene i e: Ijuana GDA 63536 N 28.560◦/W 16.172◦30 4
Can ab ian
Co nice
BER Bizkaia: Be meo GDA 63539 N 43.392◦/W 2.734◦12 5
BAK Bizkaia: Bakio GDA 65364 N 43.425◦/W 2.845◦18 5
CUN As u ias: San Es eban de Cuñaba GDA 63537 N 43.277◦/W 4.676◦30 5
EUM A Co uña: Eume GDA 63535 N 43.404◦/W 8.087◦30 5
LIE Can ab ia: Liendo GDA 65365 N 43.375◦/W 3.383◦30 5
NUE As u ias: Nue a de Llanes GDA 63538 N 43.421◦/W 4.954◦30 5
SEI A Co uña: Seixo GDA 63530 N 43.706◦/W 7.946◦19 5
GDA, Uni e si y o G anada he ba ium.
Plan s 2024,13, 1587 5 o 26
Plan s 2024, 13, x FOR PEER REVIEW 4 o 26
he effec o clonali y on i s in aspeci ic gene ic s uc u e. To quan i y he gene ic a ia-
ion and i s dis ibu ion wi hin and be ween popula ions, and o iden i y sui able a eas
o he species in he pas and he u u e, we used nuclea mic osa elli e loci, one plas id
ma ke , and species dis ibu ion modeling (SDM) wi h a double aim. Fi s ly, we sough
o in e he species’ phylogeog aphic his o y. The biogeog aphic pa e n o he species,
ypical o a elic axon, makes C. mac oca pa a good sys em o assess he impac o bo h
p e-Pleis ocene and Qua e na y geological and clima ic e en s on popula ion p ocesses
ha gene a e gene ic s uc u e, and p esen insigh s in o how ongoing and u u e clima e
change may shape e olu iona y pa e ns. Fu he mo e, phylogeog aphic da a om an-
cien axa, such as C. mac oca pa, may also p o ide insigh s in o he demise o he Palaeo-
opical lo a o Eu ope. Secondly, we sough o es ima e he impo ance o sexual and
ege a i e ep oduc ion on popula ion composi ion and o e alua e he effec o clonali y
on in aspeci ic gene ic s uc u e.
2. Ma e ials and Me hods
2.1. Plan Ma e ial
A o al o 443 indi iduals o C. mac oca pa was sampled om 17 popula ions in 4
geog aphical egions ac oss i s dis ibu ion ange: Andalusia, he Azo es, he Cana y Is-
lands, and he Can ab ian Co nice. In Madei a, whe e he species is epo edly also p e-
sen , we could no ind any popula ion. The numbe o popula ions pe egion was be-
ween one and i e, and he numbe o sampled indi iduals pe popula ion a ied om 6
o 30 (Table 1; Figu e 2).
Figu e 2. Map o he Culci a mac oca pa sampling loca ions. F equency o each mul ilocus lineage
(MLL) based on mic osa elli e da a is ep esen ed as pie cha s pe loca ion. Colo s ep esen he
diffe en MLLs ha a e sha ed among si es, and yellow ep esen s p i a e MLLs ha only appea
CRMFOG
CAR
NAT
CID EUM
SEI
IJU
BER
BAK
CUN
SDN
RM
PIN
ALM
NUE
LIE
N
30º W 20º W 0º W10º W
30º N
40º N
50º N
Azo es
Cana y Islands
A lan ic Ocean
A ica
Ibe ian
Peninsula
Madei a
0 500 1,000 Km
Figu e 2. Map o he Culci a mac oca pa sampling loca ions. F equency o each mul ilocus lineage
(MLL) based on mic osa elli e da a is ep esen ed as pie cha s pe loca ion. Colo s ep esen he
di e en MLLs ha a e sha ed among si es, and yellow ep esen s p i a e MLLs ha only appea
in one si e. See Table 1 o he ull name o loca ions. No e ha he pie cha size is he same o all
loca ions and does no ep esen he numbe o indi iduals.
2.2. DNA Ex ac ion, Mic osa elli e Geno yping, and Plas id DNA Sequencing
To al genomic DNA o he 443 indi iduals was ex ac ed om silica-d ied onds
ollowing a modi ied low-sal CTAB ex ac ion p o ocol [
40
]. We de eloped eigh mi-
c osa elli e loci, which we e used o geno ype all indi iduals (see Table S1 o mic osa el-
li e cha ac e is ics). Geno yping was pe o med on an ABI PRISM
®
3100-A an Gene ic
Analyze (Applied Biosys ems, Fos e Ci y, CA, USA). Alleles we e sco ed using GENE-
MARKER 1.85 (So Gene ics, S a e College, PA, USA).
Fo plas id DNA (p DNA) analysis, a subsample o 82 indi iduals (3–5 pe popu-
la ion) was used. The plas id ma ke used was he in e genic space ps4- nL, he only
polymo phic egion o he 13 es ed (in ons in pl16, ps16, nG
UCC
, nL, and yc 3, and
he in e genic space s pl32- nL, ps4- nL, ps16-ma K, nD-psbM, nD- poB, nH-psbA,
nL- nF, and nS
GGA
- nG
UCC
). All p ime pai s used o PCR ampli ica ion a e shown
in Table S2. PCR eac ions we e pe o med in 25
µ
L eac ions con aining 50 ng o genomic
DNA, 1.25
µ
M o each p ime , 2 mM MgCl
2
, 1.5 mM dNTPs, 2.5
µ
L Bio ools 10
×
Reac ion
Bu e , and 1.5 uni s o Bio ools DNA polyme ase (Bio ools B&M Labo a o ies S.A., Mad id,
Spain). Cycling pa ame e s consis ed o 5 min o dena u ing a 94
◦
C; ollowed by 35 cycles
o 94
◦
C o 1 min, 50
◦
C o 1 min, and 72
◦
C o 1 min; and a inal ex ension a 72
◦
C
o 10 min. Sequencing was pe o med on an ABI PRISM
®
3100-A an Gene ic Analyze
(Applied Biosys ems, Fos e Ci y, CA, USA). The esul ing sequences we e aligned by eye
using he alignmen edi o BIOEDIT 7.0.5.3 [41].
Plan s 2024,13, 1587 6 o 26
2.3. Clonali y and Gene ic Di e si y
2.3.1. Mic osa elli es
To in e he clonal iden i y o he sampling uni s (all indi iduals sampled), and o
disc imina e be ween gene ically dis inc indi iduals esul ing om di e en zygo es
(gene s) and independen clonal eplica es gene ically iden ical o he pa en plan ( ame s),
we i s ly es ed he esolu i e powe o he eigh mic osa elli e ma ke s by es ima ing
he geno ype accumula ion cu e using he unc ion geno ype_cu e om he R package
POPPR 2.8.3 [
42
]. This unc ion samples andom subse s o loci by he use o a Mon e
Ca lo p ocedu e, and examines he obus ness o he in e ed clonal membe ships. Then,
we used MLGsim 2.0 [
43
] o calcula e he p obabili y ha epea ed mul ilocus geno ypes
(MLGs) o igina ed om di e en sexual ep oduc ion e en s (p
sex
; being gene ically dis inc
indi iduals, di e en gene s). This was based on he obse ed allele equencies and
he sample size o he da a se , while aking in o accoun depa u es om he Ha dy–
Weinbe g equilib ium (HWE) when es ima ing geno ypic p obabili ies (p
gen
(F
IS
), o a
mo e conse a i e es ima e o p
sex
; [
44
]). The signi icance alues o p
sex
we e de e mined by
compa ison wi h he dis ibu ion o 1000 simula ed p
sex
alues. Finally, o de ine he clonal
lineages o mul iple locus lineages (MLLs; i.e., di e en MLGs belonging o a di e en
o he same clone), we analyzed he dis ibu ion o he equencies o gene ic dis ances
be ween pai s o MLGs, wi h he unc ion mlg. il e and using B u o dis ances on POPPR.
The gene ic h eshold dis ance unde which wo MLGs we e conside ed he same MLL
was es ima ed using he a hes -neighbo me hod.
The clonali y desc ip o s we e calcula ed wi h he unc ion popp on POPPR as ollows.
Fi s ly, o cha ac e ize he clonal ichness, we de e mined he numbe o MLLs, he numbe
o expec ed MLLs (eMLLs), and he clonal ichness (R) co ec ed o he sampling size.
Secondly, o cha ac e ize he geno ype di e si y, we calcula ed he Simpson’s index (lambda;
co ec ed o sampling size) and he clonal e enness index (E.5), which shows how equally
each MLL is ep esen ed. Finally, we calcula ed he s anda dized associa ion index (
d
; [
45
])
o es he p edominan ep oduc i e model (sexual, whe e linkage among loci is no
expec ed, s. clonal, whe e signi ican disequilib ium is expec ed due o linkage among
loci). This la e index was also es ima ed co ec ing o clones and hen using only one
indi idual pe MLL, in o de o es he e ec o pa ial clonali y. The signi icance o
d
was
es ed wi h a pe mu a ion es (10,000 pe mu a ions). All desc ip o s we e es ima ed bo h
a he popula ion and geog aphical egion le els.
To calcula e gene ic di e si y desc ip o s, we used all sampling uni s (including
ame s), ollowing he ecommenda ion o [
46
], because his way, he eal gene ic s uc u e
o C. mac oca pa popula ions is mo e accu a ely ep esen ed. Thus, we calcula ed: he
numbe o alleles (A) and allelic ichness (A ), a e ac ing o he smalles sample size, using
he R package HIERFSTAT 0.04-30 [
47
] wi h he unc ions allele.coun and allelic. ichness,
espec i ely; he obse ed and expec ed he e ozygosi y (H
O
and H
E
, espec i ely), and
ixa ion index F
IS
and HWE, using GENODIVE 3.0 [
48
]. Fu he mo e, o in es iga e how
he de ini ion o he indi idual ha we used ( ame -based s. gene -based de ini ions)
in luences he es ima es o in a-popula ion gene ic di e si y indices (as ecommended
by [
49
]), we also calcula ed gene ic desc ip o s (A ,H
O
,H
E
, and F
IS
) using only one
indi idual pe MLL pe popula ion (only gene s). S uden ’s es s we e pe o med o
explo e signi ican di e ences be ween da a se s (including s. excluding clones). All
gene ic desc ip o s we e calcula ed a he popula ion and geog aphical egion le els.
2.3.2. p DNA
Gene ic di e si y was assessed by he numbe o haplo ypes (ha), haplo ype di e si y
(Hd), and nucleo ide di e si y (
π
) calcula ed using ARLEQUIN 3.5.2.2 [
50
]. All di e si y
indices we e calcula ed a he popula ion and geog aphical egion le els.
Plan s 2024,13, 1587 7 o 26
2.4. Gene ic S uc u e and Phylogeog aphy
S anda d and hie a chical analyses o molecula a iance (AMOVA; [
51
]) we e used o
es o pa i ioning o gene ic a iabili y wi hin samples, wi hin and be ween popula ions,
and be ween he ou geog aphical egions. Fo mic osa elli es, hese analyses we e made
wi h all sampling uni s (including ame s) and wi h only one indi idual pe MLL pe popu-
la ion (gene s), using he unc ion popp .amo a on POPPR and he unc ion and es , unning
1000 eplica es, o es o signi icance. Fo p DNA, we used he p og am ARLEQUIN,
and he signi icance was es ed wi h 10,000 pe mu a ions. In addi ion, one mo e AMOVA
analysis was ca ied ou o p DNA conside ing he geog aphical dis ibu ion o he haplo-
ypes and he esul o he ne wo k analysis (see he Resul s Sec ion). Thus, we included a
ou h le el o popula ion g ouping called sup a- egional g ouping. Two sup a- egional
g oups we e conside ed: Sou h (Andalusia, he Azo es, and he Cana y Islands) and he
Can ab ian Co nice.
Popula ion gene ic s uc u e was analyzed using di e en app oaches wi h ou mi-
c osa elli e da a. Fi s , pai wise F
ST
alues we e calcula ed, bo h wi h all sampling uni s
and wi h only one indi idual pe MLL pe popula ion, be ween popula ions using GEN-
ODIVE; he signi icance o F
ST
was es ed by a pe mu a ion es wi h 10,000 pe mu a ions.
We compa ed he alues ound wi h and wi hou clones using he Spea man’s co ela ion
coe icien . Second, he Bayesian algo i hm implemen ed in STRUCTURE 2.3.4 [
52
] was
used o e alua e he numbe o gene ic clus e s (K) bo h wi h all sampling uni s and wi h
clone co ec ion. The numbe o clus e s es ed anged om one o 18, wi h 10 eplica es
pe K, using he no-admix u e model and independen allele equencies. The bu n-in
pe iod and Ma ko Chain Mon e Ca lo (MCMC) i e a ions we e se o 50,000 and 10
6
,
espec i ely. The op imal numbe o clus e s was es ima ed wi h he online ool STRUC-
TURESELECTOR [53]. We iden i ied he uppe mos hie a chical le el o gene ic s uc u e
using he del a K-me hod (
∆
K; [
54
]). To explo e o he le els o gene ic pa i ioning, we
used he ou independen es ima o s p oposed by [
55
] (MedMedK, MedMeaK, MaxMedK,
and MaxMeaK), conside ing a membe ship coe icien h eshold o 0.5. To align and isual-
ize he STRUCTURE ou pu ac oss he 10 eplica es, we used he online ool CLUMPAK
1.1 [
56
]. Thi d, he gene ic s uc u e was also assessed using a model- ee mul i a ia e
s a is ics-based clus e ing me hod, a disc iminan analysis o p incipal componen s (DAPC)
on R package ADEGENET 2.1.10 [
57
] using all sampling uni s. The unc ion x alDapc
om ADEGENET was used o selec by c oss- alida ion he co ec numbe o p incipal
componen s wi h 1000 eplica es using a aining se o 90% o he da a. The numbe o
p incipal componen s was chosen based on he c i e ia ha i had o p oduce he highes
a e age pe cen age o success ul eassignmen and lowes oo mean squa ed e o [57].
The e olu iona y ela ionships and geog aphical dis ibu ion o p DNA haplo ypes
we e explo ed by econs uc ing a haplo ype ne wo k ollowing he s a is ical pa simony
me hod [58] as implemen ed in TCS 1.21 [59].
2.5. Gene Flow Using Mic osa elli e Da a
We es ed he connec i i y among popula ions by es ima ing he mig a ion a es
among hem, consis en ly wi h all sampling uni s. Thus, o de e mine whe he ecen (o e
wo o h ee gene a ions) gene low had occu ed be ween he popula ions, we es ima ed
he mig a ion a es (m) be ween all indi idual popula ions using a Bayesian assignmen es
wi h he so wa e BAYESASS 1.3 [
60
]. As p og am se ings, he de aul alues we e used
(MCMC i e a ions, 3
×
10
6
; leng h o he bu n-in, 999,999; sampling equency, 2000; del a
alue, 0.15). Isola ion by dis ance (IBD) was es ed o he 17 popula ions using he eg es-
sion o pai wise F
ST
dis ances [de e mined wi h GENODIVE using hem ans o med as
F
ST
/(1
−
F
ST
)] and loga i hms o geog aphical dis ances be ween popula ions, by applying
a Man el es in GENODIVE.
Plan s 2024,13, 1587 8 o 26
2.6. Species Dis ibu ion Modeling
Po en ial e uge and u u e dis ibu ion a eas o C. mac oca pa we e de e mined by
pe o ming species dis ibu ion modeling (SDM). This analysis equi es he p esence oc-
cu ence da a o he species s udied and en i onmen al a iables. Fo en i onmen al da a,
we used 19 BIOCLIM a iables a a esolu ion o 2.5 a c minu es (ca. 5 km) ep esen ing
di e en ime pe iods du ing pas , p esen , and u u e clima ic condi ions. Pas and cu en
clima e da a we e a ailable om he Wo ldClim da abase (www.wo ldclim.o g; [
61
]) and
included da a o he cu en -day pe iod (1950–2000), he Las Glacial Maximum (LGM; c.
21 ka) simula ed by he CCSM model ( he Communi y Clima e Sys em Model), and o
he Las In e glacial pe iod (LIG; c. 120 ka). We ob ained p edic ions o u u e clima ic
condi ions in he yea 2080 o he IPCC clima e scena io wi h he mos impac : RCP8.5 [
62
]
a ailable h ough he CCAFS Clima e po al (www.cca s-clima e.o g). Soil da a we e de-
i ed om SoilG ids.o g [
63
] bu we e no used wi h pas clima ic condi ions in he LGM
because o he lack o such maps. Highly co ela ed a iables (Pea son’s R
≥
0.8) we e
educed o eigh unco ela ed a iables (Table S3) used as p edic o s o calib a e he dis ibu-
ion models. Species-occu ence da a a e a collec ion o e e ences in da abases ( he Global
Biodi e si y In o ma ion Facili y da a po al (h p://www.gbi .o g/; accessed on 15 July
2017), he Biodi e si y da abank o he Cana y Islands (h p://www.biodi e sidadcana ias.
es/a lan is/common/index.js ; accessed on 16 June 2013), he Azo es Biodi e si y da abank
(h p://www.a lan is.ang a.uac.p /a lan is/common/index.js ; accessed 1 Feb ua y 2014),
he li e a u e [
64
–
67
], plus ou own ield eco ds. A o al o 379 p esence eco ds we e
inally compiled (Figu e S1). To pe o m he SDM, we applied maximum en opy model-
ing implemen ed in he so wa e package MAXENT 3.4.1 [
68
]. Models we e gene a ed
using a c oss- alida ion o 5 eplica e uns. Model pe o mance was assessed based on
he a ea unde he ecei e ope a ing cha ac e is ic cu e (AUC). The con ibu ion o each
p edic o a iable in he model was analyzed by he pe mu a ion impo ance and pe cen
con ibu ion coe icien s (Table S3). A inal educed model including he mos impo an
a iables [
69
], i.e., he mean diu nal ange and minimum empe a u e o coldes mon h,
was inally compu ed.
3. Resul s
3.1. Clonali y and Gene ic Di e si y
Mic osa elli es
A o al o 120 di e en mul ilocus geno ypes (MLGs) we e de ec ed among he 443 sam-
pling uni s. The geno ype accumula ion cu e showed ha ou eigh mic osa elli e loci
had s ong powe o disc imina e be ween he MLGs o C. mac oca pa, since wi h se en loci,
almos 100% o he MLGs we e esol ed (Figu e S2). Only o one epea ed MLG could we
no ule ou an independen o igin by sexual ep oduc ion (p
sex
= 0.96, p= 0.115). This MLG
was sha ed be ween wo sampling uni s o di e en popula ions, CAR ( he Azo es) and
NUE ( he Can ab ian Co nice), and he e o e hese and all he sampling uni s o di e en
popula ions wi h sha ed MLGs we e main ained in he da a se when clone co ec ion
was applied. The gene ic h eshold dis ance unde which wo MLGs we e conside ed o
belong o he same mul ilocus lineage (MLL) was 0.0391 (Figu e S3). A e he MLGs we e
collapsed in o MLLs, he o al numbe o MLLs was 104 dis ibu ed among 130 indi iduals
(gene ically dis inc indi iduals; gene s) ac oss he popula ions, wi h di e en numbe s
o clones be ween popula ions and geog aphical egions (Figu es 2and S4A,B; Table 2).
Be ween 1 and 23 MLLs we e de ec ed ac oss all he popula ions. Thi een MLLs we e
sha ed among popula ions, o which he mos equen showed a di e en ial dis ibu ion
be ween Andalusia, he Azo es, and he Can ab ian Co nice (Figu e 2, MLLs: ed, o ange,
and whi e and black). The es o he MLLs we e exclusi e o he popula ions (Figu e 2;
Table 2). The Can ab ian Co nice was he egion ha e ained he highes numbe o MLLs
(53) and clonal ichness (R = 0.31), al hough his was no e enly dis ibu ed among he
popula ions. Many Can ab ian popula ions had e y low numbe s o MLLs ( wo we e
e en monoclonal, i.e., EUM and LIE), while CUN (23 MLG; R = 0.759) and NUE (18 MLG;
Plan s 2024,13, 1587 9 o 26
R = 0.586) ha bo ed he highes alues o all popula ions sampled. On he con a y, in
he Azo es, despi e ha ing ewe MLLs (35, R = 0.29), hei popula ions ha bo ed ela-
i ely high and simila le els o clonal ichness (Table 2), and he expec ed MLL (eMLL;
Table 2) alues we e e en highe han hose o he Can ab ian Co nice. The di e en ial
p e alence o clones be ween he Azo es popula ions and be ween hose o he Can ab ian
Co nice is e lec ed in he egional e enness index (Table 2; Figu e S4A,B). Al hough he
CUN and NUE popula ions showed a p opo iona e dis ibu ion o clones and he e o e
had a high e enness alue, he dominance o a ew clones in o he popula ions (excep
BER) made he Can ab ian Co nice he egion wi h he lowes alue o e enness. The
Azo es, meanwhile, was he egion wi h he highes p opo ionali y in he dis ibu ion o
clones (excluding he Cana y Islands) and also wi h he g ea es clonal di e si y (lambda;
Table 2). Con e sely, Andalusia was he egion wi h lowes alues o clonal ichness and
geno ype di e si y, since he popula ions showed ew MLLs and only some we e dominan
(Figu es 2and S4A,B; Table 2). In he Cana y Islands, al hough he only known popula ion
p esen ed mode a e o low alues o clonal ichness, he geno ype di e si y indexes we e
close o hose ound in he Azo es and he mos di e se popula ions o he Can ab ian
Co nice (Table 2). All he popula ions o which he associa ion index could be calcula ed,
excep FOG, NAT, and IJU, and geog aphical egions, excep he Cana y Islands, showed
a signi ican linkage disequilib ium when all sampling uni s we e included. When only
one indi idual was conside ed pe MLL pe popula ion, only PIN a he popula ion le el,
and Andalusia and he Can ab ian Co nice a he egional le el, p esen ed signi ican
disequilib ium (Table 2).
In o al, 37 alleles we e de ec ed om he eigh loci su eyed. Be ween wo and i e
alleles pe locus we e ound ac oss all he popula ions (Table 3). Allelic ichness and he
expec ed he e ozygosi y (H
E
) signi ican ly changed when only one indi idual pe MLL
pe popula ion was conside ed ( = 3.05; p< 0.05 and =
−
2.69; p< 0.05 espec i ely), bu
he obse ed he e ozygosi y (H
O
) and he ixa ion index (F
IS
) did no change ( =
−
1.41;
p< 0.17 and =
−
1.04; p< 0.31, espec i ely). All popula ions, excep ALM, de ia ed om
HWE when all indi iduals we e included, esul ing in signi ican ly nega i e alues o FIS
in he popula ions CRM, RM, SDN, IJU, CUN, and LIE. When only one indi idual pe MLL
pe popula ion was conside ed, he F
IS
alues emained nega i e o hese popula ions
(excep o SDN and LIE wi h only one MLL each) (Table 3). The nega i e alues o F
IS
we e de e mined mainly by he locus CM-AT19, which showed ixed he e ozygosi y ( o
only wo alleles) in almos all popula ions, and he high numbe o monomo phic loci in
he di e en popula ions (Table S4). A e CM-AT19 was excluded, only he popula ions
RM, IJU, and CUN egis e ed nega i e F
IS
alues (signi ican only in RM). Wi h espec
o he egions, he F
IS
alues showed a signi ican excess o homozygo es (excep he
Cana y Islands, wi h only one popula ion), bo h including all sampling uni s and only
one indi idual pe MLL pe popula ion. A he popula ion le el, hose o he Azo es and
Can ab ian Co nice (especially BER, NUE, and CUN) showed he highes di e si y alues,
e sus he lowes in Andalusia; howe e , a he egional le el, Andalusia had di e si y
le els simila o hose o he o he egions (Table 3).
When conside ing he p e alence o ege a i e p opaga ion in he popula ions (clonal
ichness, R; Figu e S5), on he one hand, we ound an upwa d end in he alues o allelic
ichness (A ) and he expec ed he e ozygosi y (H
E
) wi h dec easing clonali y (highe R).
Howe e , no clea e ec o clonali y on obse ed he e ozygosi y (H
O
) was disce ned, and
popula ions wi h highe clonal p e alence in gene al had F
IS
alues a he om ze o. On
he o he hand, clonali y appea ed o in luence he ex en o he di e ences be ween he
es ima es o he di e si y indices when applying he di e en de ini ions o “indi idual”
(conside ing clonal eplica es, ame s, s. including only gene ically dis inc indi iduals,
gene s). Thus, wi h he inc ease in clonali y, he di e ences o he es ima es o H
O
,H
E
,
and F
IS
inc eased, while o A he di e ences we e g ea e he less clonal (g ea e R) he
popula ions we e (Figu e S5). In he case o F
IS
, he di e ences we e g ea e because, when
Plan s 2024,13, 1587 16 o 26
he con idence in e al when he da a o e no in o ma ion) signi y no cu en gene low
be ween he popula ions.
The Man el es , wi h and wi hou clones, indica ed a lack o isola ion by dis ance
ac oss he popula ions ( = 0.076, p= 0.209 wi h clones; = 0.055, p= 0.304 wi hou clones).
3.4. Species Dis ibu ion Modeling
Fo all he models, he AUC alues we e high (minimum alue o AUC = 0.990). The
MAXENT cu en and LIG p edic ions showed egions o sui able habi a s ha coincided
la gely wi h he species’ cu en dis ibu ion, wi h addi ional a eas o i s dis ibu ion ange
in he Eu opean A lan ic coas s u he no h and he Medi e anean Sea, whe e he species
is cu en ly absen (Figu es 6and S8). Acco ding o LGM ou pu s, e ugia we e loca ed in
Maca onesia, he coas o Po ugal, Galicia (whe e C. mac oca pa is cu en ly p esen ), and
he Eu opean A lan ic coas (a la i udes o he p esen no h o F ance and sou h o G ea
B i ain, whe e he species is cu en ly absen ). Palaeodis ibu ion modeling sugges ed
no sui able habi a s o C. mac oca pa on he no he n coas o Spain (excep in Galicia)
whe e he species is cu en ly ound. The MAXENT u u e p ojec ions (yea 2080) using he
RCP8.5 scena io sugges ed a pa ial educ ion in sui able habi a s on he coas s o Po ugal,
no he n Ibe ian Peninsula, and Maca onesian islands oge he wi h an inc ease in sui able
habi a s no hwa d o he Eu opean A lan ic coas .
Plan s 2024, 13, x FOR PEER REVIEW 16 o 26
ange in he Eu opean A lan ic coas s u he no h and he Medi e anean Sea, whe e he
species is cu en ly absen (Figu es 6 and S8). Acco ding o LGM ou pu s, e ugia we e
loca ed in Maca onesia, he coas o Po ugal, Galicia (whe e C. mac oca pa is cu en ly
p esen ), and he Eu opean A lan ic coas (a la i udes o he p esen no h o F ance and
sou h o G ea B i ain, whe e he species is cu en ly absen ). Palaeodis ibu ion modeling
sugges ed no sui able habi a s o C. mac oca pa on he no he n coas o Spain (excep in
Galicia) whe e he species is cu en ly ound. The MAXENT u u e p ojec ions (yea 2080)
using he RCP8.5 scena io sugges ed a pa ial educ ion in sui able habi a s on he coas s
o Po ugal, no he n Ibe ian Peninsula, and Maca onesian islands oge he wi h an in-
c ease in sui able habi a s no hwa d o he Eu opean A lan ic coas .
Figu e 6. Po en ial dis ibu ion o Culci a mac oca pa d awn wi h MAXENT 3.4.1. (Top le ), a he
p esen ime (Cu en ); ( op igh ), a he Las In e glacial (LIG, ca. 120,000 yea s BP); (bo om le ),
a he Las Glacial Maximum (LGM, ca. 21,000 yea s BP) using he Communi y Clima e Sys em
Model (CCSM); (bo om igh ), p edic ion o he yea 2080 unde RCP 8.5 condi ions.
4. Discussion
Culci a mac oca pa has been ega ded as one o he e ns o he he baceous laye o
Eu opean lau oid o es s du ing he Te ia y and one ha , a e he geological–clima ic
e en s o he Miocene and Pliocene, su i ed in Maca onesian and Ibe ian shel e s [5].
Cu en ly, due o he educed and agmen ed na u e o i s dis ibu ion a ea, C. mac oca pa
is conside ed a species unde h ea and p o ec ed unde a ious Eu opean, Po uguese,
and Spanish p o ec ion ca ego ies [29–33]. In his s udy, we elucida e he phylogeog aphic
his o y o C. mac oca pa and assessed he impac o ege a i e ep oduc ion on popula ion
dynamics and gene ic a iabili y. In addi ion, we e alua e how he de ini ion o “indi id-
ual” chosen ( ame -based, conside ing all sampling uni s, including clonal eplica es, s.
gene -based, conside ing only gene ically dis inc indi iduals excluding clonal eplica es)
affec s es ima es o in a-popula ion gene ic di e si y indices and gene ic s uc u ing.
Azo es
Madei a
Cana y Isl.
N
Cu en
0 500 1,000 Km
0 – 0.2
0.2 – 0.4
0.4 – 0.6
0.6 – 0.8
0.8 – 1
Azo es
Madei a
Cana y Isl.
N
LIG
0 500 1,000 Km
0 – 0.2
0.2 – 0.4
0.4 – 0.6
0.6 – 0.8
0.8 – 1
Azo es
Madei a
Cana y Isl.
N
LGM
0 500 1,000 Km
0 – 0.2
0.2 – 0.4
0.4 – 0.6
0.6 – 0.8
0.8 – 1
Azo es
Madei a
Cana y Isl.
N
2080
0 500 1,000 Km
0 – 0.2
0.2 – 0.4
0.4 – 0.6
0.6 – 0.8
0.8 – 1
0º10º W20º W30º W 10º E0º10º W20º W30º W 10º E
40º N
30º N
50º N
60º N
40º N
30º N
50º N
60º N
40º
N
30º
N
50º
N
60º
N
40º
N
30º
N
50º
N
60º
N
0º10º W20º W30º W 10º E0º10º W20º W30º W 10º E
Figu e 6. Po en ial dis ibu ion o Culci a mac oca pa d awn wi h MAXENT 3.4.1. (Top le ), a he
p esen ime (Cu en ); ( op igh ), a he Las In e glacial (LIG, ca. 120,000 yea s BP); (bo om le ), a
he Las Glacial Maximum (LGM, ca. 21,000 yea s BP) using he Communi y Clima e Sys em Model
(CCSM); (bo om igh ), p edic ion o he yea 2080 unde RCP 8.5 condi ions.
Plan s 2024,13, 1587 17 o 26
4. Discussion
Culci a mac oca pa has been ega ded as one o he e ns o he he baceous laye o
Eu opean lau oid o es s du ing he Te ia y and one ha , a e he geological–clima ic
e en s o he Miocene and Pliocene, su i ed in Maca onesian and Ibe ian shel e s [
5
].
Cu en ly, due o he educed and agmen ed na u e o i s dis ibu ion a ea, C. mac oca pa
is conside ed a species unde h ea and p o ec ed unde a ious Eu opean, Po uguese,
and Spanish p o ec ion ca ego ies [
29
–
33
]. In his s udy, we elucida e he phylogeog aphic
his o y o C. mac oca pa and assessed he impac o ege a i e ep oduc ion on popula ion
dynamics and gene ic a iabili y. In addi ion, we e alua e how he de ini ion o “indi idual”
chosen ( ame -based, conside ing all sampling uni s, including clonal eplica es, s. gene -
based, conside ing only gene ically dis inc indi iduals excluding clonal eplica es) a ec s
es ima es o in a-popula ion gene ic di e si y indices and gene ic s uc u ing.
4.1. Clonali y E ec on Gene ic Di e si y
The eigh newly iden i ied mic osa elli e loci had s ong powe o disc imina e be ween
he mul ilocus geno ypes o C. mac oca pa, demons a ing hei use ulness as ma ke s o
s udying clonali y and gene ic di e si y in his species. The alues o he clonal desc ip o s
disco e ed in he C. mac oca pa popula ions analyzed (Table 2) and AMOVA analyses
(wi h he highes p opo ion o di e si y ound wi hin samples, only when one MLL
pe indi idual pe popula ion was conside ed; Table 4) con i m he subs an ial o e all
clonali y o his species. Al hough clonali y was de ec ed in all popula ions, i did no a ec
hem equally. In gene al, a highe p e alence o clonali y was ound in Andalusian and
Can ab ian popula ions (excep h ee). Consequen ly, he impac o clonali y on gene ic
di e si y le els a ied be ween popula ions. The highe he clonal p e alence, he lowe
he H
E
and A (Figu e S5). Acco ding o p e ious esea ch [
23
], he dec ease in hese
pa ame e s in clonal popula ions may esul om he absence o meiosis and ecombina ion
o in e clonal compe i ion (leading o he elimina ion o less-adap ed clones) dec easing he
amoun and equency o alleles. Howe e , in popula ions ending owa d s ic clonali y,
clones will accumula e he e ozygosi y o e ime h ough mu a ion e en s a each locus,
leading o high he e ozygo e excess, wi h each locus, in ini e popula ions, becoming ixed
o a he e ozygous s a e [
24
]. Among he C. mac oca pa popula ions ha we e s ic ly clonal
o ha had e y high clonal p e alence, many (i.e., CRM, IJU, LIE, RM, and SDN) had
H
O
alues (al hough low) much highe han H
E
, esul ing in high he e ozygo e excess
( e y nega i e F
IS
alues). On he con a y, popula ions such as BAK, EUM, and SEI
we e almos en i ely o en i ely homozygous, de ia ing om he expec ed pa e n o
s ic ly clonal popula ions. Fu he mo e, only one locus showed ixed he e ozygosi y in
almos all popula ions, he majo i y being homozygous o mos popula ions. The obse ed
pa e n in mo e clonal popula ions o C. mac oca pa appea s o be bes explained by he
species’ b eeding sys em, whe e in a-game ophy ic sel ing appea s o be a o ed [
35
].
In a-game ophy ic sel ing, an ex eme o m o inb eeding, in diploid e ns p oduces
homozygous spo ophy es a all loci. Fu he mo e, clonali y inc eases sel - e iliza ion
a es, con ibu ing o he gene ic impo e ishmen o popula ions [
78
], also explaining he
dec easing le els o gene ic di e si y in popula ions wi h inc easing clonali y.
Ou esul s indica e ha clonali y a ec s he es ima ed alues o gene ic di e si y
and s uc u ing pa ame e s, in ag eemen wi h p e ious au ho s [
49
], who highligh ed
he isk o misin e p e ing hese pa ame e s, depending on he de ini ion o “indi idual”
adop ed ( ame -based s. gene -based). The abo e s udy [
49
] ocused on H
E
a ia ion,
e ealing ha highe clonali y leads o g ea e di e ences be ween H
E
es ima es o ame s
and gene s in a popula ion. Acco ding o hese au ho s, he ex en and di ec ion o his
di e ence will depend on he size dis ibu ion o he geno ypes (% o o al ame s) and
whe he he clonal geno ype is he e ozygous o homozygous a he locus. In C. mac oca pa
popula ions, we obse ed he pa e n desc ibed in he abo e s udy, wi h a ia ions be ween
H
E
es ima es inc easing wi h highe clonali y. In all cases, ame -based H
E
was lowe han
Plan s 2024,13, 1587 18 o 26
gene -based H
E
, aligning wi h he expec ed di ec ion o he expansion o a homozygous
clone acco ding o [49]. This is consis en wi h he p edominan homozygosi y a ou loci.
Conce ning allelic ichness (A ), he obse ed pa e n is he opposi e o H
E
, wi h
smalle di e ences be ween es ima es wi h highe clonali y, and consis en ly highe ame -
based es ima es compa ed o gene -based es ima es. A alues depend hea ily on he
numbe o genes conside ed o a e ac ion (2
×
no. o indi iduals in he popula ion wi h
he ewes indi iduals; g alue in [
79
]). As gdec eases, A subs an ially educes. In he
case o clonal species such as C. mac oca pa, popula ions wi h e y ew gene s will esul in
a signi ican educ ion in gwhen he gene -based app oach is employed o calcula e A ,
educing i o wo in cases o monoclonal popula ions, as obse ed in ou s udy. This ac o
de e mined he di ec ion o he a ia ion be ween es ima es ( ame -based A > gene -based
A ). On he o he hand, he smalle magni ude in he a ia ion be ween es ima es wi h
inc easing clonali y is in luenced by he e ec o he dec ease in he numbe o genes
analyzed pe popula ion (Nin [
79
]) and he dis ibu ion o alleles among indi iduals.
Gene ally, dec eases in N(by elimina ion o ame s) lead o inc eases in A , as occu ences
o he mos - equen alleles diminish wi h educed clonal edundancy, while he numbe o
occu ences o less- equen alleles a ies minimally. The ewe alleles a locus has and he
mo e homozygous i is (as occu s in mo e clonal popula ions), he mo e p opo ional he
educ ion in occu ences o he mos - equen alleles will be o he dec ease in Nand he
less he occu ences o he less- equen alleles will a y. These changes will esul in highe
A alues, because he lowe he N, he mo e he less- equen alleles will con ibu e o he
allelic ichness o a locus. The con ibu ion o he mos - equen alleles ha dly a ies ( he
educ ion in bo h is p opo ional). Thus, in clonal popula ions, whe e a smalle numbe o
alleles and g ea e homozygosi y (due o he highe sel ing a e), and a smalle numbe
o di e en mul ilocus geno ypes (gene s) a e expec ed, es ima ing A based on gene s
will subs an ially dec ease Ncompa ed o he ame -based app oach (less p onounced in
less clonal popula ions). Consequen ly, A ends o inc ease, pa ly compensa ing o he
dec ease in i s alue due o he smalle numbe o genes used o a e ac ion (g). As a
esul , he di e ence be ween ame -based and gene -based A es ima es is smalle in mo e
clonal popula ions. In he ex eme cases o clonali y (i.e., CRM, EUM, LIE, and SDN), he
obse ed a ia ion was minimal o non-exis en .
Rega ding H
O
and F
IS
alues, di e ences be ween es ima es based on he de ini ion o
“indi idual” we e no ed p ima ily in he mos clonal popula ions. In hese popula ions, he
emo al o clonal edundancy esul ed in a highe p opo ion o he e ozygous indi iduals
(excep in he IJU and RM popula ions wi h high H
O
) and co ec ion o he F
IS
alue owa d
ha o a sexual popula ion.
Ou esul s indica e a s ong in e -popula ion di e en ia ion in C. mac oca pa. The-
o e ical p edic ions sugges ha , in clonal o ganisms wi h a sexual ep oduc ion a e,
clonal ep oduc ion ends o augmen di e en ia ion be ween popula ions compa ed o
he pa en al popula ion. This is due o he endency o in a-popula ion H
E
o dec ease
wi hou a co esponding inc ease in o al H
E
, leading o highe F
ST
alues. Howe e , in
cases o s ic clonali y, popula ions end o show less di e en ia ion han sexual o gan-
isms because clonali y p e en s allele ixa ion [
80
]. In he case o C. mac oca pa, e en he
mos s ic ly clonal popula ions exhibi ed e y high le els o di e en ia ion and s ongly
homozygous loci, con a y o expec a ions o s ic ly clonal popula ions. Thus, in C. mac o-
ca pa, he high le els o popula ion di e en ia ion appea o esul om he combined
e ec o sel ing and clonali y. As men ioned ea lie , sel ing inc eases homozygosi y, lead-
ing o allele ixa ion and popula ion di e en ia ion, while clonali y enhances he e ec
o sel ing by boos ing he a e o sel - e iliza ion. Al hough he esul s ob ained o ge-
ne ic s uc u e wi h ame -based and gene -based de ini ions we e gene ally cong uen ,
he gene -based app oach was less obus o de ec ing gene ic s uc u e in C. mac oca pa,
showing lowe le els o in e -popula ion di e en ia ion by elimina ing clonal edundancy
(Tables 4and S5; Figu e S6).
Plan s 2024,13, 1587 19 o 26
4.2. Phylogeog aphy and Popula ion Dynamics
Due o he abili y o e ns o dispe se o e long dis ances, hei popula ions ha e been
cha ac e ized as ha ing low gene ic di e en ia ion, wi h mos o he a ia ion occu ing a
he in a-popula ion le el [
10
]. Con a y o his, ou esul s show a global phylogeog aphy
o C. mac oca pa cha ac e ized by he di e en ia ion o popula ions in wo main g oups,
which coincide wi h he geog aphical dis ibu ion o he species in he no he n Ibe ian
Peninsula as well as in he Maca onesian a chipelagos and he sou he n Ibe ian Peninsula.
In addi ion o his o e a ching model, we ha e also de ec ed a s ong in e -popula ion
di e en ia ion, e en wi hin each o he wo main g oups, wi h he absence o ecen gene
low. The assessmen o he con ibu ion o indi idual alleles o popula ion s uc u ing,
acili a ed by DAPC analysis (Figu e S7), enabled us o elucida e hose ha played a majo
ole in dis inguishing he wo popula ion g oups and, u he mo e, o p o ide e idence
o he exis ence o dispe sal p ocesses, e en be ween hese g oups, as indica ed by he
dis ibu ion pa e n o hese alleles ac oss popula ions. A simila phylogeog aphic model
has been desc ibed o Vandenboschia speciosa (Willd.) G. Kunkel (Hymenophyllaceae),
a Te ia y species wi h a dis ibu ion simila o ha o C. mac oca pa (al hough mo e
widesp ead no hwa d along he Eu opean A lan ic coas and owa d cen al Eu ope),
whose popula ions a e s uc u ed in wo e olu iona y uni s, one om he no h ( om
he Can ab ian Co nice o he no h and cen al Eu ope) and ano he om he sou h
(Maca onesia, Andalusia, and I aly) [19].
In eg a ing he esul s o gene ic di e si y, p e alence o clonal ep oduc ion, and
popula ion di e en ia ion pain s a mo e complex pic u e han wo dis inc e uge egions
o C. mac oca pa. I sugges s ha , wi hin each p e iously p oposed sup a- egional g oup
( he sou he n g oup and he Can ab ian Co nice), some popula ions o egions migh ha e
unc ioned as e ugia, while o he s appea o be he esul o pos -glacial dispe sal e en s.
A key expec a ion o glacial e ugia is ha popula ions pe sis ing he e o ex ended
pe iods will ha bo highe gene ic di e si y compa ed o ecolonizing popula ions. These
la e popula ions a e usually composed o subse s o he gene ic di e si y p esen in he
sou ce e ugial popula ion and ypically unde go ounde e ec s and bo lenecks, culmi-
na ing in educed gene ic di e si y. Addi ionally, p olonged isola ion be ween popula ions
in sepa a e e ugia should lead o gene ic di e en ia ion due o gene ic d i [81].
In he p esen s udy, as expec ed o e ugia, popula ions in he Azo es, and CUN
and NUE ( he Can ab ian Co nice) showed high ela i e gene ic di e si y alues (Table 3;
Figu e S5), low clonali y (Figu es 2and S4A,B; Table 2), and low in e -popula ion di e -
en ia ion (Figu es 3,4and S6; Tables S5 and S6). This suppo s he cha ac e iza ion o
he Azo es ( he sou he n sup a- egional g oup) and CUN/NUE ( he Can ab ian Co nice
sup a- egional g oup) as e uges, a leas du ing he Las Glacial Maximum. By con-
as , he esul s o Andalusian popula ions and o he Can ab ian Co nice popula ions
ollow he expec a ions o pos -glacial dispe sal e en s. These popula ions exhibi ed
low gene ic di e si y (Table 3) and high clonali y wi h a ew dominan clonal lineages
(Figu es 2and S4A,B; Table 2). They also showed clea popula ion di e en ia ion
(Figu es 3,4and S6; Tables S5 and S6), e en among geog aphically close popula ions
(e.g., CRM and RM a a 2 km dis ance) o be ween he wo in a-popula ion nuclei o PIN.
This model o low gene ic di e si y and high popula ion di e en ia ion has been demon-
s a ed in popula ions esul ing om dispe sal e en s in se e al species o ock-dwelling
e ns (e.g., [21,82,83]).
A p e ailing assump ion is ha island popula ions exhibi lowe gene ic di e si y
compa ed o hei mainland coun e pa s. Howe e , ou da a om he wo a chipelagos
s udied do no suppo his no ion. Ins ead, ou analyses e ealed ha island popula ions
show le els o gene ic and geno ype di e si y simila o o highe han hose o mainland
popula ions (Tables 2and 3). Ou indings add o he g owing body o e idence demon-
s a ing ha island popula ions a e no inhe en ly less di e se ( e iewed by [
84
]), bu a he
ha o he ac o s need o be conside ed. Fo ins ance, as in he case o C. mac oca pa, he
ole o islands as clima ic e ugia (whe e popula ions pe sis o e long pe iods) and he
Plan s 2024,13, 1587 20 o 26
abili y o some plan species o dispe se hei p opagules o e long dis ances, as expec ed
o spo e-p oducing plan s, ( acili a ing mul iple coloniza ion wa es and hus seconda y
con ac and hyb idiza ion o lineages) a o la ge, e ec i e popula ion sizes in island
popula ions, which ha e been shown o be mo e gene ically di e se han hei mainland
coun e pa s, and which can unc ion as mig a o y s epping s ones and can e en ecolonize
he mainland (e.g., [19,85,86]).
The p esen esul s o species dis ibu ion modeling (SDM) sugges high sui abili y
o C. mac oca pa in he Azo es du ing he Las In e glacial (LIG) and he Las Glacial
Maximum (LGM), suppo ing he idea o he a chipelago se ing as a glacial e uge o
he species (Figu es 6and S8). Howe e , SDM esul s do no suppo he p oposal (null
sui abili y) ha mos o he Can ab ian Co nice (including CUN and NUE) ac s as a glacial
e uge, bu a he he da a sugges high sui abili y in he mos no h-wes e ly end o he
Ibe ian Peninsula (Galician coas ) and he Po uguese coas (Figu es 6and S8). These
esul s sugges a ecoloniza ion o he Can ab ian Co nice om hose places du ing he
Holocene. F om a gene ic s andpoin , i his we e ue, we should expec he Galician
popula ions o ha e he highes gene ic di e si y in he Can ab ian Co nice, wi h di e si y
dec easing eas wa d ollowing he ecoloniza ion di ec ion (isola ion by dis ance model).
Howe e , ou esul s show ex emely low gene ic di e si y in Galician popula ions and,
in addi ion, no e idence was ound o isola ion by dis ance. Ou esul s o he Galician
popula ions align wi h hose o a p e ious s udy using isozymes, which epo ed a single
mul ilocus geno ype ac oss all indi iduals o m six popula ions [
38
]. To econcile he
SDM and gene ic di e si y esul s, whe e CUN and NUE appea o be he mos di e se
popula ions, we specula e ha in he Can ab ian Co nice, popula ions migh ha e pe sis ed
in small, clima ically a o able pocke s isola ed om he p e ailing clima ic condi ions.
These popula ions could ha e hen se ed as sou ces o pos -glacial ecoloniza ion o he
Can ab ian Co nice in a ious di ec ions. A simila pa e n o di e si y dis ibu ion and
habi a sui abili y in he Can ab ian Co nice was obse ed o he elic e n Vandenboschia
speciosa [19].
The cha ac e is ics o C. mac oca pa popula ions wi h dispe sal signa u es a e con-
sis en wi h s ong bo lenecks due o ecen ounde e en s by one o ew geno ypes,
ollowed by expansion h ough ege a i e ep oduc ion. This phenomenon is known in
clonal he baceous plan s (e.g., [
87
,
88
]) and pa icula ly in e ns, whe e mos homospo e
e ns a e belie ed o es ablish popula ions om a single spo e ia in a-game ophy ic
sel ing [
9
,
82
,
89
]. Fo example, his coloniza ion s a egy (single-spo e coloniza ion and
subsequen popula ion es ablishmen ) was p oposed o he pos -glacial coloniza ion o
Eu ope by Asplenium ichomanes subsp. quad i alens D. E. Mey [21].
This coloniza ion s a egy esul s in a comple ely homozygous spo ophy e de i ed
om a single haploid game ophy e. In a-game ophy ic sel ing, due o he esul ing ho-
mozygosi y, is p edic ed o ha e nega i e e olu iona y consequences [
90
]. I educes
gene ic a ia ion wi hin lineages, limi ing hei abili y o adap o changing en i onmen s.
Addi ionally, inc eased homozygosi y can expose pa ially ecessi e dele e ious mu a ions,
leading o dec eased e ili y and lowe su i al [
90
]. The e o e, o ully exploi he coloniza-
ion ad an age o e ed by in a-game ophy ic sel ing, species mus mi iga e hese nega i e
e ec s. Polyploidy, wi h i s addi ional genome(s), has been p oposed as a mechanism ha
may bu e agains he nega i e gene ic consequences o in a-game ophy ic sel ing. The
addi ional genomes po en ially p e en o delay he exposu e o dele e ious mu a ions
o selec ion, allowing popula ions o pe sis e en wi h sel ing. Al e na i ely, i has been
shown ha in aspeci ic a ia ion in ma ing sys ems migh be widesp ead, wi h geno ypes
in isola ed popula ions exhibi ing he highes sel ing capaci y ega dless o ploidy le el [
91
].
The la e sugges s selec ion o geno ypes mo e ole an o he de imen al e ec s o
in a-game ophy ic sel ing. These ole an geno ypes would likely be he ones in ol ed
in coloniza ion p ocesses. Following coloniza ion, subsequen sexual ep oduc ion wi h
addi ional immig an geno ypes is necessa y o achie e inc eased gene ic di e si y and
iabili y, especially o ou c ossing diploids [
90
]. In his con ex , ege a i e p opaga ion
Plan s 2024,13, 1587 21 o 26
and/o spo ophy ic sel ing [
91
] could allow popula ion expansion while p olonging he
ime window o he a i al o new immig an s. This s a egy could explain he high
di e si y obse ed in long-li ed popula ions, such as hose in he Azo es.
The highly homozygous popula ions EUM and BAK, and popula ions wi h nea -
comple e homozygosi y a e excluding locus CM-AT19 (wi h ixed he e ozygosi y), could
be examples o coloniza ion om a single spo e ollowed by es ablishmen h ough in a-
game ophy ic sel ing and ege a i e ep oduc ion. Howe e , hese popula ions likely ha e
no had su icien ime o ecei e enough immig an s o inc ease hei gene ic di e si y.
Ou esul s a e consis en wi h cul u e expe imen s demons a ing ha C. mac oca pa ga-
me ophy es a e ini ially male bu la e become he maph odi ic [
34
,
35
]. This, coupled wi h
he lack o an an he idiogen sys em in C. mac ocap a, appea s o a o in a-game ophy ic
sel ing [
36
,
37
]. This suppo s he no ion ha selec ion o sel ing geno ypes may occu du -
ing long-dis ance coloniza ion e en s. The high homozygosi y and linkage disequilib ium
(based on he associa ion index (Table 2)) de ec ed in ounded popula ions o C. mac oca pa
compa ed o sou ce popula ions u he sugges di e en ial sel ing capaci y, co obo a ing
he idea aised by p e ious esea che s [91].
The analysis o gene ic di e si y s uc u e and he dis ibu ion pa e n o alleles wi h
he g ea es con ibu ion o his s uc u e, iden i ied h ough DAPC analysis, p o ide e i-
dence o he occu ence o long-dis ance dispe sal e en s be ween he wo sup a- egional
g oups iden i ied in C. mac oca pa. The STRUCTURE analysis, conside ing all sampling
uni s (Figu e 3), e ealed a hi d clus e linking some Can ab ian Co nice popula ions
(BER, CUN, and NUE) wi h wo Andalusian popula ions (RM and PIN) and he Cana y
Island popula ion (IJU). This ela ionship was also e lec ed by he F
ST
alues and DAPC
esul s (Figu es 4and S7; Table S5). No ably, in he DAPC analysis, Andalusian and Cana y
Island popula ions sha ed alleles wi h high- esolu ion alues o indi idual assignmen o
Can ab ian popula ions (i.e., CM35_106, AT9_267, CM1A_209, and AT30_194; Figu e S7). In
addi ion, al hough he wo main plas id DNA (p DNA) haplo ypes (H-I and H-II) exhibi a
di e en ia ed geog aphical dis ibu ion, wi h H-I cha ac e izing he Maca onesian g oup
and H-II cha ac e izing he Can ab ian g oup, occasional excep ions a ose. The p esence
o H-I in he Can ab ian Co nice and H-II in Andalusia and he Azo es implies disc e e
long-dis ance dispe sal e en s.
The subs uc u e de ec ed wi hin Andalusia and he con as ing nuclea and plas id
a ini ies o i s popula ions compa ed o o he egions sugges mul iple coloniza ion e en s
o his egion, po en ially om he Azo es and he Can ab ian Co nice (o om cu en ly
ex inc popula ions geog aphically close o Andalusia). While he RM popula ion and one
in a-popula ion nucleus o PIN sha e mic osa elli e p o iles wi h CUN and NUE popu-
la ions, hey possess he ypical p DNA haplo ype o he Maca onesian g oup. Howe e ,
he p esence o his p DNA haplo ype also in CUN and NUE sugges s a long-dis ance
dispe sal e en owa d Andalusia, ollowed by local dispe sal om RM o PIN o ice
e sa. No ably, PIN and RM sha e he only p i a e haplo ype ound in Andalusia, u he
suppo ing he con en ion o local dispe sal.
The p esence o H-II in he ALM popula ion sugges s ano he long-dis ance dispe sal
e en , po en ially o igina ing om he Can ab ian Co nice o he Azo es (Figu e 5A).
The la e seems mo e likely, gi en he nuclea ela ionship o ALM wi h he Azo es and
o he Andalusian popula ions in he STRUCTURE analysis (pa icula ly a he uppe mos
hie a chical le els, K= 2 and K= 3; Figu e 3) and he DAPC analysis based on Disc iminan
Func ion 1 (Figu e 4). Simila esul s we e obse ed in Diplazium cauda um (Ca .) Je my [
86
],
whe e i s p esence in Andalusia (i s only mainland locali y) was a ibu ed o ecoloniza ion
a e a long-dis ance dispe sal e en om Maca onesia (p esumably he Cana y Islands)
wi h subsequen es ablishmen by a single spo e o ew spo es h ough in a-game ophy ic
sel ing and ege a i e p opaga ion, ollowed by local dispe sals.
In he Cana y Islands, only one popula ion o C. mac oca pa is known. This popula ion,
wi h in e media e le els o gene ic di e si y and clonali y (Tables 2and 3), could ep esen
a e uge popula ion. Al e na i ely, i migh be a ecen ly es ablished popula ion, wi h
Plan s 2024,13, 1587 22 o 26
ela i ely high he e ozygosi y esul ing om he accumula ion o soma ic mu a ions and/o
addi ional immig an geno ypes. The limi ed expansion wi hin he a chipelago compa ed
o o he elic e ns sha ing simila habi a s (e.g., Diplazium cauda um,P e is incomple a Ca .,
and Vandenboschia speciosa) sugges s a ecen a i al, al hough ou da a canno de ini i ely
con i m his.
A limi a ion o his s udy is he lack o samples om Madei a, which would p o ide a
mo e comple e unde s anding o he connec ions be ween egions wi hin he Maca onesian
g oup. In a p e ious s udy on Vandenboschia speciosa, Madei a was ound o be closely
ela ed o he sou he n Ibe ian Peninsula, while he Azo es we e associa ed wi h he
no he n e olu iona y uni [
19
]. Fo C. mac oca pa, he Azo es popula ions clea ly ela e
o he sou h o he Ibe ian Peninsula and he Cana y Islands. The absence o samples
om Madei a hinde s ou abili y o e alua e i s po en ial ole in acili a ing he connec ion
be ween he Azo es, he Cana y Islands, and he sou he n Ibe ian Peninsula.
4.3. Conse a ion and Fu u e Pe spec i es
The iden i ica ion o wo sup a- egional g oups ( he Can ab ian co nice/Sou h) could
help design a managemen plan o imp o e conse a ion measu es. This e n is especially
h ea ened in Andalusia, whe e only haplo ypes and alleles a e p esen , making i c i ical
o p o ec his a ea. In ela ion o p edic ions, SDM esul s in 2080 show a hea y loss o
habi a sui abili y o C. mac oca pa in Andalusia and in he Cana y Islands, and also o he
no h-wes e n Ibe ian Peninsula, bu a gain in habi a sui abili y owa d no he n Eu ope
(Figu es 6and S8). All his wa ns o a po en ial h ea o he popula ions o he sou he n
g oup and implies a no hwa d mig a ion o he species, necessi a ing he p o ec ion o he
sou he n popula ions in an e o o p e en he loss o gene ic di e si y.
The esul s ega ding he a ia ion in he es ima es o gene ic di e si y and gene ic
s uc u ing desc ip o s, depending on he adop ed de ini ion o “indi idual”, emphasize
he need o cau ion in in e p e ing he es ima es when o mula ing managemen and
conse a ion measu es o endange ed species wi h clonal p opaga ion. The choice be ween
gene -based and ame -based de ini ions can esul in an unde - o o e -es ima ion o hese
pa ame e s. Following he ecommenda ion o o he au ho s [
49
], in cases whe e he
ege a i e p opaga ion o he species is known, pa ame e s should be adop ed using
bo h app oaches.
Supplemen a y Ma e ials: The ollowing suppo ing in o ma ion can be downloaded a : h ps://
www.mdpi.com/a icle/10.3390/plan s13121587/s1, Table S1: Cha ac e is ics o eigh mic osa elli e
loci de eloped in Culci a mac oca pa; Table S2: P ime pai s used o PCR ampli ica ion o he
13 p DNA egions es ed in Culci a mac oca pa; Table S3: Pe cen age con ibu ion and pe mu a ion
impo ance (MaxEn ) o selec ed model o he species dis ibu ion modeling (SDM); Table S4: Values
o F
IS
pe popula ion and pe locus wi h and wi hou locus CM-AT19; Table S5: Pai wise popula ion
F
ST
o mic osa elli es; Table S6: Mean ecen mig a ion a es (m) among he s udied popula ions,
es ima ed om eigh mic osa elli e loci using he BAYESASS p og am; Figu e S1: Loca ion o p esence
eco ds used o species dis ibu ion modeling (SDM); Figu e S2: Geno ypic accumula ion cu e
showing he esolu i e powe o he eigh mic osa elli es used in his s udy; Figu e S3: His og am
o equency dis ibu ion o pai wise gene ic dis ances; Figu e S4: Dis ibu ion o he 104 MLLs
among he 130 indi iduals (gene s) o Culci a mac oca pa; Figu e S5: Sca e plo s o gene ic di e si y
es ima es, ob ained wi h eigh mic osa elli e loci, agains clonal ichness (R) o Culci a mac oca pa
popula ions; Figu e S6: Ba plo s showing he STRUCTURE esul s, using mic osa elli e da a and
assuming he no-admix u e model; Figu e S7: Addi ional esul s o he disc iminan analysis o
p incipal componen s (DAPC); Figu e S8: De ailed po en ial dis ibu ion o Culci a mac oca pa in
he Ibe ian Peninsula and Maca onesian Islands d awn wi h MAXENT. Re s. [92,93] a e ci ed in he
Supplemen a y Ma e ials.
Au ho Con ibu ions: Concep ualiza ion, V.N.S.-S.; Fo mal analysis, V.N.S.-S., J.P. and S.B.-M.S.;
Me hodology, V.N.S.-S. and S.B.-M.S.; P ojec adminis a ion, V.N.S.-S.; Supe ision, V.N.S.-S.;
W i ing—o iginal d a , V.N.S.-S. and S.B.-M.S.; W i ing— e iew and edi ing, V.N.S.-S., J.P. and
A.T.R.-G. All au ho s ha e ead and ag eed o he published e sion o he manusc ip .
Plan s 2024,13, 1587 23 o 26
Funding: This esea ch ecei ed no ex e nal unding.
Da a A ailabili y S a emen : All sequence da a ob ained in his s udy a e a ailable om he Gen-
Bank da abase (mic osa elli e accession numbe s: OR965870–OR965877; p DNA accession numbe s:
OR965866–OR965869).
Acknowledgmen s: Sami a Ben-Menni Schule was g an ed a p edoc o al g an (F.P.U. p og am)
om he Spanish Go e nmen . The au ho s hank all hose people and ins i u ions ha acili a ed o
helped in he collec ion o samples (in alphabe ical o de : Amado P ie o Fe nández, Ángel Baña es,
An onio Delgado, Elizabe h Ojeda, Gab iel Blanca, Gobie no de Cana ias, Jun a de Andalucía, and
Miguel Pé ez-Gu ié ez).
Con lic s o In e es : The au ho s decla e no con lic s o in e es .
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