Supporting Information
Journal of Biogeography
Evidence for multiple founding lineages and genetic admixture in the evolution
of species within an oceanic island weevil (Coleoptera, Curculionidae) superradiation
Christiana M. A. Faria, Antonio Machado, Isabel R. Amorim, Matthew J. G. Gage,
Paulo A.V. Borges and Brent C. Emerson
Appendix S1
Table S1 - List of sampling localities of the Laparocerus tessellatus complex in the
Canary Islands with corresponding XY geographic coordinates.
Code
LP1
LP2
LP3
LP4
LP6
LP7
EH9
EH10
EH11
TF12
TF13
TF14
TF15
TF20
TF21
TF22
TF23
TF24
TF25
TF27
TF28
TF43
TF45
TF46
Locality
La Palma: Montaña de Tagoja
La Palma: Llanada de Barlovento
La Palma: s. El Paso
La Palma: Breña Alta
La Palma: Mazo: Venijobre
La Palma: El Paso: Montaña de Don Mendo
El Hierro: Monte Ajares
El Hierro: San Andrés: Piedras Blancas
El Hierro: Cruz de Isora
Tenerife: Cumbres de Arico
Tenerife: Granadilla: Las Vegas
Tenerife: Arico: Contador
Tenerife: Tanque Bajo
Tenerife: Cumbre de Bolicos
Tenerife: s. Montaña Bermeja
Tenerife: Güímar: Barranco Del Agua
Tenerife: s. Icod El Alto
Tenerife: Santa Úrsula: Barranco de Bensa
Tenerife: Tacoronte: Fuente Fría
Tenerife: Santa Úrsula: La Corujera
Tenerife: El Portillo
Tenerife: Pista de Las Yedras
Tenerife: Anaga: El Pijaral Km 4.5
Tenerife: Anaga: Chinobre
X
28.724580
28.817258
28.652309
28.619452
28.608606
28.561485
27.803063
27.756203
27.743302
28.197701
28.145034
28.191434
28.361465
28.313392
28.327162
28.308071
28.360967
28.390079
28.420114
28.403039
28.302921
28.538693
28.555583
28.558559
Y
-17.782751
-17.809252
-17.845581
-17.822510
-17.786075
-17.859593
-17.917333
-17.965891
-17.978965
-16.531152
-16.545207
-16.527426
-16.775051
-16.820181
-16.533197
-16.442346
-16.598623
-16.454661
-16.407498
-16.492287
-16.566531
-16.300133
-16.181180
-16.175192
Table S1 (cont.)
Code
TF47
TF48
TF49
GC31
GC32
GC34
GC35
GC36
GC37
GC39
GC40
GC41
GC42
Locality
Tenerife: Anaga: Cruz del Carmen
Tenerife: Las Raíces
Tenerife: Ifonche
Gran Canaria: Valsendero: Barranco Oscuro
Gran Canaria: Tamadaba NW
Gran Canaria: Barranco de los Cernícalos
Gran Canaria: San Bartolomé
Gran Canaria: San Bartolomé
Gran Canaria: Degollada de Osorio
Gran Canaria: Cumbre: Roque Redondo
Gran Canaria: Valsendero
Gran Canaria: Las Huertecillas
Gran Canaria: Barranco de la Mina
X
28.531925
28.423824
28.132672
28.067702
28.053239
27.959703
27.912163
27.926115
28.072543
28.089959
28.043199
28.090170
27.998256
Y
-16.280070
-16.379237
-16.688040
-15.588936
-15.691922
-15.531192
-15.572533
-15.577780
-15.557942
-15.593852
-15.593991
-15.560884
-15.587043
Details of PCR amplification and sequencing of the two genes used to investigate
evolutionary history of the Laparocerus tessellatus complex in the Canary Islands
A fragment of approximately 785 bp of the mitochondrial gene cytochrome c oxidase
subunit II (COII) was amplified using primers TL2-J-3038 (5’TAATATGGCAGATTAGTGCATTGGA) (Emerson et al., 2000) and TK-N 3782
(5’-GAGACCATTACTTGCTTTCAGTCATCT) (EVA-Harrison Laboratory, Cornell
University, Ithaca, NY, USA). Primers M13REV-CAS5p8sFt (5’CAGGAAACAGCTATGACCTGAACATCGACATTTYGAACGCATAT) (Ji et al.,
2003; as modified in Regier & Shi, 2005) and CAS28sB1d (5’TTCTTTTCCTTCSCTTAYTRATATGCTTAA) (Ji et al., 2003) were used to
amplify a fragment of approximately 540 bp of the nuclear gene internal transcribed
spacer 2 (ITS2).
Polymerase chain reactions (PCR) contained NH4 buffer (1x), 3.0 mM MgCl2 (for
COII reactions and 4.0 mM MgCl2 for ITS2 reactions), 0.2 mM of each dNTP, 0.4
µM of each primer and 0.5 U of Taq polymerase (Bioline) in 25µL final volume. PCR
cycles were carried out using the following thermal profile for COII: 95 ºC for 3 min,
32 cycles at 95 ºC for 1 min, annealing temperature 48 ºC for 1 min, 72 ºC for 1 min,
and a final extension at 72 oC for 3 min. For ITS2 the thermal profile used was: 94 ºC
for 3 min, 34 cycles at 95 ºC for 40 sec, annealing temperature 50 ºC for 1 min, 72 ºC
for 40 sec, and a final extension at 72 ºC for 2 min. Sequencing was performed in a
PerkinElmer ABI3700 automated sequencer with BigDyeTerminator v3.1 Cycle
Sequencing kit (applied Biosystems, California, USA). The thermal profile used for
all sequencing reactions was: 96 ºC for 10 sec, 50 ºC for 5 sec and 60 ºC for 4 min, 25
cycles. Sequences for COII were obtained with the forward primer only. The majority
of COII PCR products yielded clean and unambiguous sequences with the forward
primer (TL2-J-3038), but in some samples the first 48 nucleotides were not clear,
requiring the use of an internal reverse primer (LapCOIIRev1 5’GGYATRAATCTATGATTTGTT), which binds at positions 588 to 608 at the end 3’
of the aligned sequences. COII and ITS sequences used in the present study are
available for download at http://ncbi.nlm.nih.gov under GenBank accession numbers
KT275683-KT275810.
Appendix S2
Simulation of stochastic lineage sorting
To evaluate stochastic lineage sorting as an explanation for shared mitochondrial
genetic variation between La Palma and the islands of Gran Canaria and Tenerife,
simulations of lineage extinction were performed for Gran Canaria and Tenerife.
Contemporary mtDNA sequence variation sampled from the island of Tenerife
consists of 28 different mtDNA haplotypes, with 26 on the island of Gran Canaria.
Considering the close relatedness of some haplotypes from Tenerife and La Palma
(Figure 2, node D), we allow for the possibility that closely related mtDNA
haplotypes within the islands of Tenerife and Gran Canaria could have evolved
subsequent to a colonisation event from either island to La Palma. We thus consider a
combined ancestral pool of 47 haplotypes shared across both islands by removing 7
closely related haplotypes from the data set. We simulated the ancestral occurrence of
shared variation between Gran Canaria and Tenerife such that all 47 haplotypes occur
on both islands, yielding 94 geographically references haplotypes. We then simulated
the random extinction of 50% of these 94 haplotype records, with 100 simulations, to
estimate haplotype sharing between Gran Canaria and Tenerife from incomplete
lineage sorting. After 100 simulated extinction events, an average of 24 shared
haplotypes (range: 16-30 haplotypes) were estimated between islands. The minimum
estimate of 16 shared haplotypes does not support an explanation of incomplete
lineage sorting for the observed data, where no closely related sequences are shared
between the islands of Gran Canaria and Tenerife.
Analysis of species boundaries in Tenerife
The joint analysis of mitochondrial and nuclear markers provides the opportunity to
assess the biological significance of divergent genetic lineages when those lineages
are sampled in sympatry (e.g. Cicconardi et al., 2013). Divergent lineages can be
formally evaluated for consistency with the biological species concept (BSC) (Mayr,
1942) by testing for Hardy–Weinberg equilibrium (HWE) and linkage disequilibrium
(LD) when they occur sympatrically within sampling sites. In the case of divergent
genetic lineages sampled sympatrically within species of the L. tessellatus complex,
HWE and LD were calculated using ARLEQUIN 3.5.1.2 (Excoffier & Lischer, 2010),
and statistical significance was tested using 100,000 dememorization steps and
100,000 steps in Markov chain, with Bonferroni adjustments (Benjamini & Hochberg,
1995) for multiple comparisons. The null hypothesis considered that individuals
within a sampling site belong to a panmictic population. Heterozygote deficit (FIS) at
the ITS locus within populations sampled for divergent mtDNA lineages was assessed
using FSTAT (Goudet, 1995). Similar analyses were also conducted for other
populations with sufficiently large sample size, but for which only a single mtDNA
lineage was sampled.
Divergent mitochondrial lineages occurring sympatrically were found at two sampling
sites in Tenerife (Fig. 2, sites TF45 and TF48) and the possibility of cryptic species
was evaluated by assessing co-segregation of alleles (e.g. Cicconardi et al., 2013).
The genotypes sampled at these sites are listed in Table S2. For the 11 individuals
(successfully sequenced for ITS2) of L. tessellatus sampled at site TF45,
mitochondrial lineages correspond to different nuclear groupings, while at site TF48
(n=11), individuals of L. freyi with divergent mitochondrial lineages are consistent
with a single nuclear grouping. At site TF45, both null hypotheses of linkage
equilibrium and HWE were rejected (p-adj<0.001), while at site TF48 neither of the
null hypotheses could be rejected (p>0.05).
Heterozygote deficit (FIS) at the ITS locus within populations was calculated for sites
T45 and T48, as well as three other sites with sufficiently large sample sizes (LP6,
TF22 and GC35). Sites TF45, TF48 and GC35 presented high levels of heterozygote
deficit (FIS: 0.48, 0.46 and 0.65 respectively) while the populations TF22 and LP29
presented lower FIS values (0.12 and 0.06 respectively). All FIS values were
significantly positive (p-adj=0.004).
Table S2 - Genotypes of individuals collected sympatrically in two sites in Tenerife
(TF45 – El Pijaral and TF48 – Las Raíces). Both sites are represented by only one of
the two species, but each presents sequence variation from two divergent mtDNA
lineages found on Tenerife (Figs. 2 and 3). MtDNA alleles are preceded by “C” and
nuclear alleles are preceded by “IT”. Individual L157 sampled from TF45 is not
included, as ITS2 sequences were not obtained for this individual.
TF45 (El Pirajal)
Individual
COII
lineage
TF48 (Las Raíces)
Genotype
Individual
COII
lineage
Genotype
150
C76
IT20/29
165
C76
IT24/24
153
C76
IT51/51
167
C76
IT06/06
C76
IT51/51
169
C76
IT24/24
154
Clade 2
Clade 2
155
C76
IT20/52
172
C76
IT24/24
156
C76
IT20/52
174
C76
IT24/24
158
C76
IT51/51
175
C76
IT24/24
148
C25
IT06/24
166
C22
IT20/24
151
C29
IT20/28
171
C22
IT24/24
C29
IT29/29
173
C23
IT24/24
147
C33
IT06/06
170
C24
IT20/24
149
C33
IT06/06
168
C28
IT24/24
152
Clade 4
Clade 4
Appendix S3
Figure S1
Bayesian phylogenetic tree of the complete mtDNA COII data set (172 sequences, 633 bp)
inferred using the GTR+G model of sequence evolution showing relationships within the
Canary Islands Laparocerus tessellatus complex. The tree is rooted with L. vicinus. Bayesian
posterior probabilities are shown below nodes. Letters and numbers immediately to the right
of species names correspond to location codes, see Table 1.
Figure S2
Bayesian phylogenetic tree of the complete ITS2 nuclear gene dataset (330 sequences, 411
bp) inferred using the GTR+G model of sequence evolution showing relationships within the
Canary Islands Laparocerus tessellatus complex. The tree is rooted with L. vicinus. Bayesian
posterior probabilities are shown below nodes. Letters and numbers immediately to the right
of species names correspond to location codes, see Table 1.
Figure S1 (part 1)
Figure S1 (part 2)
Figure S2 (part 1)
Figure S2 (part 2)
Figure S2 (part 3)
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