ARTICLE IN PRESS
Journal of
Arid
Environments
Journal of Arid Environments 66 (2006) 477–497
www.elsevier.com/locate/jnlabr/yjare
Using statistical phylogeography to infer population
history: Case studies on Pimelia darkling beetles
from the Canary Islands
Ó. Moyaa, H.G. Contreras-Dı́aza,b, P. Oromı́b, C. Juana,
a
Departament de Biologia, Universitat de les Illes Balears, 07122 Palma de Mallorca, Spain
Departamento de Biologı´a Animal, Facultad de Biologı´a, Universidad de La Laguna, 38205 La Laguna,
Tenerife, Spain
b
Available online 3 March 2006
Abstract
Sequence data from a 200 bp fragment of the Cytochrome Oxidase I mitochondrial gene was
derived from endemic populations of the darkling beetle Pimelia laevigata (Coleoptera,
Tenebrionidae) from the volcanic islands of La Gomera, La Palma and El Hierro and from three
related congeneric species of Tenerife (Canary Islands). Statistical phylogeographic methods and
estimates of demographic parameters suggest that there is a higher genetic variation and
geographical structure in two of the Tenerife nominal species than in populations of P. laevigata
in the western islands. In La Gomera, La Palma and El Hierro, the patterns are consistent with
relatively recent colonizations, followed by range expansions. The results show that hypotheses based
on coalescent theory can be useful to reconstruct historic biogeographical events of oceanic islands in
a range of different organisms provided that the sample design is adequate and enough genetic
resolution is present. However, some specific problems arise when interpreting the inference key
applied to the volcanic islands populations.
r 2006 Elsevier Ltd. All rights reserved.
Keywords: Mitochondrial DNA; Nested clade phylogeographic analysis; Colonization; Population expansion;
Arid regions; Darkling beetles
Corresponding author. Tel.: +34 971173425; fax: +34 971173184.
E-mail address: [email protected] (C. Juan).
0140-1963/$ - see front matter r 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jaridenv.2006.01.008
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1. Introduction
The application of phylogenetic methods to understand the extent of genetic variation in
space and time among populations or related taxa has emerged as a major issue in
evolutionary and conservation biology. Different theoretical approaches exist in relation
to this topic, but the more common and recent uses coalescent theory (Kingman, 1982a, b)
as a conceptual framework to estimate different population parameters and understand
demographic histories (see Emerson et al., 2001; Knowles, 2004). The inferences obtained
by these methods are based on statistical tests of historical hypotheses and estimates of
demographic parameters, in contrast with the ‘‘classical’’ intuitive phylogeographic
methodology developed by Avise (1998), which is based on inferring the causes of an
association between observed patterns of genetic variation and the geographical
distribution of populations (Knowles, 2004). Nested clade phylogeographic analysis
(NCPA) is one of the statistical approaches applied to intraspecific or closely related
species gene genealogies. It combines genealogical data through haplotype networks
obtained using statistical parsimony and geographical information to infer historical range
distribution of haplotypes and range movements (Templeton et al., 1995; Templeton, 1998,
2004; Posada and Crandall, 2001).
The Hawaiian, Galápagos and Canary volcanic island chains have become models in
evolutionary studies, as they are true microcosms of evolutionary processes (Emerson,
2002). Recently, the ease in obtaining DNA sequence data, and the improvement
of phylogenetic methods, have promoted their application to many different speciose
endemic organisms from the Canary and other Macaronesian archipelagos, including
vertebrate and arthropod groups (e.g. Juan et al., 1995, 1998, 2000; Brown and
Pestano, 1998; Emerson et al., 1999, 2000a–c; Carranza et al., 2001; Rees et al.,
2001a, b; Contreras-Dı́az et al., 2003). The molecular phylogenies provide a hypothesis
of relationships of taxa, that can be used for testing the mono- or polyphyly of a particular
group of taxa on the islands, understanding colonization sequences, disentangling
extinction, hybridization and lineage sorting effects, or even comparing island speciation rates with the corresponding continental relatives (Emerson, 2002). Several studies
have used Canary Island beetles and mitochondrial DNA sequences at the population
or the species level, to study the geographical distribution of genetic diversity
(phylogeography). They generally have found consistencies with the known geographical
volcanic evolution of the island chain or different volcanic regions within a single island.
For example, two clear divergent mitochondrial lineages in Pimelia (Coleoptera,
Tenebrionidae) from Tenerife were deduced to relate to expansion from the older isolated
areas of the island (Juan et al., 1996). In the Fuerteventura and Lanzarote endemic
darkling beetle Hegeter deyrollei (formerly H. politus), a sequential expansion of
the populations concomitant with the cessation of volcanism was inferred (Juan et al.,
1998). Emerson et al. (2000a) showed for the weevil Brachyderes rugatus that the
mitochondrial haplotypes of populations from the four subspecies occurring in Gran
Canaria, Tenerife, La Palma and El Hierro belong to two different monophyletic clades
and suggested a recent origin and a possible colonization sequence for this group in the
Canary Islands.
Darkling beetles (Coleoptera, Tenebrionidae) constitute important elements of the arid
and semi-arid terrestrial ecosystems, because of their high biomass and role as detritivores
(De los Santos et al., 2000, 2002a, b). They show adaptations such as diurnal and seasonal
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rhythms of activity, resistance to desiccation and adaptation to high temperatures and
ultra-violet light (Cloudsley-Thompson, 1964) as well as tolerance to low temperatures on
high mountains (Ottesen and Sømme, 1987). The study of genetic diversity within darkling
beetle species and populations can help not only to understand adaptive trends in these
insects in relation to environmental factors, but also to give a historic perspective to the
biogeography of these key organisms in arid/semi-arid environments. For example, in a
previous study we analysed the phylogeography of the Pimelia endemic species of the
island of Gran Canaria (Contreras-Dı́az et al., 2003) and the results helped to establish
evolutionary units for conservation purposes.
Here we have studied populations of Pimelia species endemic to four islands of the
Canary archipelago. New samples of three species (Pimelia canariensis, P. ascendens and
P. radula) occurring on Tenerife are added to a previous mitochondrial sequence data set
published elsewhere (Juan et al., 1996). Using these more extensive population and species
samples, a re-analysis is performed using new phylogeographic approaches (Templeton,
2004). Pimelia canariensis is included in the List of Threatened Species in the Canaries as a
‘‘species sensitive to habitat disturbance’’. We also analyse populations of P. laevigata, a
species endemic to the three westernmost Canary islands, which according to
immunological and numerical taxonomy studies (Oromı́, 1979) and in agreement with a
previous morphological taxonomy (Español, 1961) includes three different subspecies.
Pimelia l. ssp. validipes is present in La Gomera (another Pimelia species occurs in this
island but it derives from an independent colonization event, see Contreras-Dı́az et al.,
2003), Pimelia l. ssp. laevigata in La Palma and P. l. ssp. costipennis in El Hierro. The three
subspecies of P. laevigata are broadly distributed on their correspondent islands, occurring
from arid and semi-arid lowland environments to high altitude alpine dry areas, but always
avoiding forests. In a previous study based on mitochondrial DNA sequences, the three
P. laevigata subspecies were shown to form a monophyletic group apparently derived from
Tenerife ancestors, following the east to west and older to younger island general
colonization pattern (Juan et al., 1995). The Canary Islands are of independent volcanic
origin, so colonization has to be performed across the deep waters separating them.
Although we do not have a priori knowledge of when colonization of La Gomera took
place, this island has been available for a much longer period to support founders than the
two other islands. La Gomera has been estimated to be of a maximum age about 10 million
years ago (Ma), while La Palma has been dated at 2 Ma and El Hierro less than 1 Ma
(Ancochea et al., 1990; Fuster et al., 1993; Carracedo et al., 1998).
We have sampled P. laevigata populations on the above three islands, to include a
representation of the entire geographic range of the species, and we obtained DNA
sequences for a short fragment of about 200 bp of Cytochrome Oxidase I (COI)
mitochondrial gene. This fragment showed enough variation to resolve the major
mitochondrial lineages of closely related species and populations of Tenerifean Pimelia in a
previous study (Juan et al., 1996), so its use here allows a comparison of P. laevigata
sequence diversity with that previously obtained for their Tenerife relatives. The main
questions we want to address are: (i) What is the genetic differentiation among P. laevigata
subspecies? (ii) Is there geographical structuring among P. laevigata populations within
each of the three islands where they occur? and (iii) Can NCPA and demographic
inferences help to elucidate the population history processes of relatively recent colonizers,
such as the diversification of Pimelia beetles in Tenerife and their relation to western
Canary Islands taxa?
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2. Material and methods
2.1. Sampling
Beetles were collected in Tenerife, La Gomera, La Palma and El Hierro islands of the
Canary archipelago. Fig. 1 shows the locations and codes of the samples obtained in this
study along with those sampled by Juan et al. (1996). Details of collection localities,
geographic co-ordinates, haplotype denominations and sample sizes are given in Appendix
A. Samples were stored in absolute ethanol until DNA extractions were performed.
2.2. DNA extractions, PCR amplifications and sequencing
A leg or head from each specimen was used for DNA purification with a standard
protocol (see Contreras-Dı́az et al., 2003). In some cases, the DNeasy Tissue extraction kit
(Qiagen) was used following the manufacturer’s recommendations. Pellets were
resuspended in 25–50 mL of Tris-EDTA buffer and 1 mL of the dilution was used for
PCR amplification of the fragment of COI with one unit of Taq DNA polymerase
(Ecogen). The primers used were: (50 -ACAGGAATTAAAGTTTTTAGATGATTAGC-30 )
and (50 -ATAGGGGGAATCAGTGAACTAGTCC-30 ) (Juan et al., 1996). PCR conditions
Fig. 1. Map indicating the sampling design and the areas successfully surveyed in Tenerife, La Gomera, La Palma
and El Hierro. Codes included in rectangles correspond to samples obtained in Tenerife in the previous study
(Juan et al., 1996). Symbols refer to the different species/subspecies as follows: filled squares P. ascendens, empty
squares P. canariensis, empty circle P. r. radula, filled circle P. r. oromii, and filled triangles for P. laevigata. The
population codes correspond to the ones listed in Appendix A.
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were as follows: 4 min at 95 1C followed by 35 cycles of denaturation at 95 1C for 30 s,
annealing at 50 1C for 1 min, and extension at 72 1C for 1 min, with a final single extra
extension step at 72 1C for 10 min. PCR products were checked in a 1% agarose gel and the
products of the expected length were precipitated with ammonium acetate 5 M and
isopropanol. The forward, and reverse strands in the cases where sequence ambiguities were
detected, were cycle-sequenced using an Applied Biosystems ABI Prism DYE Terminator
Cycle Sequencing Reaction Kit and sequenced in an ABI 377 automated sequencer.
A common fragment of about 200 bp was sequenced for all samples. An alignment of all the
sequences used in this study has been deposited in the EMBL database under accession
number ALIGN-000819.
2.3. Phylogenetic analyses
Sequences were aligned using the ClustalX program (Thompson et al., 1997) and no
length differences were found in the alignments. ModelTest (version. 3.06; Posada and
Crandall, 1998) was used to select the substitution model(s) that best described the data
under the Akaike information criterion, and PAUP* vs. 4.0b10 (Swofford, 2002) was used
to calculate sequence divergences and obtain phylogenetic trees. Neighbour-joining (NJ)
and maximum parsimony trees were obtained, the latter using heuristic searches with treebisection-reconnection as the branch-swapping algorithm and the starting tree was
obtained via stepwise addition with random addition of sequences with 100 replicates. The
sequence from P. lutaria, endemic to Fuerteventura and basal to all congeneric Canarian
species, as was demonstrated by using different mitochondrial and nuclear markers (Pons
et al., 2004), was used as outgroup. Bootstrap values were estimated with heuristic searches
using 500 pseudo-replicates.
2.4. Molecular diversity and population dynamics
Estimates of mean nucleotide and haplotype diversities within each species and
populations were obtained with the program DnaSP vs. 4.0 (Rozas et al., 2003). Mismatch
analysis of COI mitochondrial sequences (frequency of pairwise differences between
haplotypes) was performed to explore the demographic history of the studied populations.
This method is based on the assumption that population growth or decline leave distinctive
signatures in the DNA sequences compared to constant population size. A recent growth is
expected to generate a unimodal distribution of pairwise differences between sequences
(Rogers and Harpending, 1992). The distribution is compared to that expected under a
model of population expansion (Rogers, 1995) calculating the estimator of the time of
expansion (t) and the mutation parameter (y) according to Schneider and Excoffier (1999).
The formula t ¼ t=ð2uÞ is used to estimate the timing of population expansions. We
assume a substitution rate per site per lineage of 0.0107 per million years (equivalent to a
pairwise divergence of 2.15%/Ma assumed to be the average for insect mitochondrial
DNA) (De Salle et al., 1987; Brower, 1994), so the mutation rate u in our 200 bp COI
sequence is 2.15 106 per generation, assuming a generation time of 1 year. Confidence
intervals for the parameters of the distributions were obtained by parametric bootstrap
(1000 replicates) using Arlequin vs. 2.000 (Schneider et al., 2000). If population growth
applies, the validity of a stepwise expansion model is tested using the same bootstrap
approach by a goodness-of-fit statistic (P), representing the probability that the variance of
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the simulated data set is equal or greater than the observed data set. We also computed the
raggedness index (r) of the distribution and its significance, as implemented in Arlequin vs.
2.000. The mismatch analysis has been shown to be very conservative, having a low
statistical power in the case of low sample sizes (Ramos-Onsins and Rozas, 2002). For this
reason, other tests have been proposed for detecting past population growth such as Fu’s
F (Fu, 1997) or R2 which have been shown to be superior for small sample sizes (RamosOnsins and Rozas, 2002). For this reason, we also computed Fu’s test of neutrality as
implemented in Arlequin vs. 2.000 (Schneider et al., 2000).
2.5. Network estimation and nested clade phylogeographic analysis
The network of mitochondrial Pimelia haplotypes was inferred using statistical
parsimony (Templeton et al., 1992) as implemented in the program TCS vs. 1.13 (Clement
et al., 2000). The method links haplotypes with the smallest number of differences as
defined by a 95% confidence criterion. NCPA was used (Templeton et al., 1995) to infer
the population history of studied species of Pimelia. The NCPA first tests the null
hypothesis of no association between haplotype variation and geography, and then
proceeds to interpret the significant association patterns (Crandall and Templeton, 1993).
The NCPA nesting design was constructed by hand using the statistical parsimony
network following the rules given in Templeton (1998). In essence, the nesting procedure
consists of nesting n-step clades or haplotype groups, where n is correlated with the number
of nucleotide mutations separating haplotypes (Crandall, 1996). The procedure begins
from the external (tip) clades and proceeds to the interior (clades joined to one or more
clades by a single step). The haplotypes are nested in increasing step-levels until all the data
become nested into a single clade, the total cladogram. The software GeoDis vs. 2.1
(Posada et al., 2000) was used to calculate the NCPA distance measures and their
statistical significance. This method uses geographical distances between the sampled
locations and estimates four basic statistics: Dc, Dn, IT-Dc and IT-Dn. Dc or clade
distance measures the average distance of all clade members from its geographical centre of
distribution. Dn or nested clade distance measures how widespread is a particular clade
relative to the distribution of its sister clades in the same nesting group. IT-Dc and IT-Dn
constitute similar distances considering tips and interiors differentially. The distinction
between tip (with only one connection to the remaining network) and interior (with two
or more connections) haplotype groups in the context of the coalescent theory allows
testing the hypothesis of random geographical distribution by permutational tests
(we performed 10,000 permutations). The predictions from the coalescent can be
summarized as follows: (i) on average, haplotypes with higher frequency tend to be
older and have a greater probability of being interior, (ii) older haplotypes will be more
broadly geographically distributed, (iii) haplotypes with greater frequency will tend to have
more mutational connections and (iv) unique haplotypes (singletons) are more likely to
be joined to non-unique ones than to other singletons, and to haplotypes from the
same population (Posada and Crandall, 2001). Using the updated version of the inference
key in Templeton (2004) we can deduce which factor(s) could plausibly account for
the spatial and/or temporal significant association of haplotypes. In this way, we
can distinguish historical (fragmentation, range expansion) from current (gene flow,
genetic drift) processes responsible for the observed pattern of genetic variation
(Templeton, 2004).
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3. Results
3.1. Genetic diversity
For this study, 32 localities were sampled in the four western Canary Islands and 143
individuals have been analysed (see Fig. 1, Table 1 and Appendix A). COI sequences were
obtained for 129 individuals of P. laevigata from 27 localities across the three islands where
this species occurs (La Gomera, La Palma and El Hierro). From the sampling made in
Tenerife, we obtained 14 further individuals of P. radula and P. canariensis to complete the
geographical range of the 61 samples already sequenced in the previous study (see Fig. 1 in
Juan et al., 1996). These 75 individuals are now a good representation of the three different
nominal species present in Tenerife, one of which is split into two subspecies. Considering
that a taxonomy redefinition has been recently proposed, the haplotypes have been
arranged differently to the denominations used by Juan et al. (1996) (see Viñolas, 1994;
Oromı́ and Garcı́a, 1995).
We obtained 83 different haplotypes in our total Pimelia data set (excluding sites with
missing data) that showed considerable genetic diversity. Of those, 36 were exclusive to
Tenerife beetles and the remaining 47 belonged to P. laevigata from the westernmost
islands. Table 1 shows molecular diversity estimates for the sequence data obtained across
all Pimelia samples within Tenerife, within each of the Tenerife species, and within the
three different island populations of P. laevigata. Haplotype diversities are lower within
P. canariensis (Tenerife) (a fact that could be due to the lower sample size in this case), and
in P. l. costipennis (El Hierro) and P. l. laevigata (La Palma), suggesting a relatively recent
diversification of these populations.
3.2. Phylogenetic analyses
Overall, 60 variable positions are present in the ingroup sequence data set (of which 47
are parsimoniously informative). Sequences are AT-biased (65.8%); with most changes
Table 1
Summary of mean nucleotide and haplotype diversities of the different Pimelia species sequenced for the
mitochondrial COI fragment
Species/populations
N
Haplotypes
Nucleotide diversity7S.D.
Haplotype diversity7S.D.
P. ascendens
P. canariensis
P. radula
Total Tenerife
La Gomera
P. l. validipes
La Palma
P. l. laevigata
El Hierro
P. l. costipennis
34
8
33
75
21
2
13
36
0.017070.0020
0.005870.0013
0.026470.0038
0.051170.0019
0.91170.042
0.53670.123
0.77770.069
0.93370.017
47
21
0.012670.0019
0.84370.049
44
15
0.008770.0012
0.79570.057
38
11
0.008070.0010
0.83570.047
Total P. laevigata
129
47
0.027870.0008
0.94270.010
Total
204
83
0.053370.0018
0.96870.005
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Fig. 2. Fifty per cent majority rule consensus phylogenetic topology of the 29 maximum parsimony trees
(CI ¼ 0.322; RI ¼ 0.836) obtained based on the COI mitochondrial DNA sequence data set. Bootstrap support
(first number) of 500 pseudo-replicates and Bremer support values (second number) are indicated for the relevant
nodes. Nodes discussed in the text are labelled with letters (A and B) and numbers. Symbols at the terminal nodes
refer to the different species/subspecies as indicated in Fig. 1 from which the haplotypes were obtained.
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corresponding to synonymous substitutions at third codon positions (81.7%). The
consensus maximum parsimony tree obtained shows the haplotypes arranged into two
major sister clades (Fig. 2). Clade A includes haplotypes of P. r. oromii (north-east
Tenerife) and P. r. radula (north Tenerife), and clade B is formed by P. ascendens (west and
central regions), P. canariensis (southern arid coastal areas) plus P. laevigata
mitochondrial haplotypes from the three western islands. Haplotypes of P. r. radula from
La Matanza (MT in Tenerife) are present in clades A1 and A2. In addition, P. canariensis
haplotypes are polyphyletic with P. ascendens haplotypes, despite being the former
markedly differentiated in morphology from the high altitude P. ascendens populations
(see Español, 1961). In fact, P. ascendens had been considered just a subspecies of P. radula
when only morphological characters were used (Español, 1961); later immunological
studies showed higher differences between the two taxa and suggested their independent
species status (Oromı́, 1979), which was finally established (Oromı́ and Garcı́a, 1995) after
better supported differences based on mtDNA sequences (Juan et al., 1995). Increased
sampling of this coastal species and the use of additional molecular markers could clarify if
P. canariensis represents an ecotype of P. ascendens. Within clade B, the phylogenetic
relationships are not well resolved—what is not surprising given the short DNA sequence
used and the relatively low divergence observed.
Uncorrected distances between different Pimelia species from the easter and central
Canary Islands haplotypes ranged from 0.005 to 0.14, and the mean distance between
ingroup taxa and the outgroup P. lutaria was 0.12 (0.09–0.16 range). The optimal
substitution model for the complete data set (including the outgroup) is the General Time
Reversible (GTR, Rodrı́guez et al., 1990) with a proportion of invariable sites (I) of 0.595
and a value for the G distribution of 1.002 using the Akaike information criterion. The NJ
topology using these distances and parameters was similar to the one obtained by
maximum parsimony. A likelihood ratio test using the likelihood scores of the original tree
with branch lengths estimated implementing the GTR+I+G model assuming no
molecular clock and constraining rate constancy indicated that the data is consistent
with a molecular clock (2DL ¼ 120:88 at df ¼ 110; p ¼ 0:22).
3.3. Demographic inferences
The frequency of pairwise differences between haplotypes of each species within
Tenerife, or island populations of P. laevigata, showed mismatch distributions consisting
of unimodal curves (except for P. radula that shows two modal peaks, Figs. 3a–e). Both the
variance (SSD) and raggedness index (r) tests suggested that the curves do not significantly
differ from the distributions under the model of population expansions (PSSD ¼ 0:1820:94
and Pr ¼ 0:1720:91, see legend to Fig. 3) in accordance with the corresponding Fu’s FS
tests. The P. radula mismatch distribution shows two waves of expansion (Fig. 3b),
possibly corresponding to the geographically structured clades A1 and A2. Accordingly,
the more stringent Fu’s test of neutrality (Fu, 1997) gave a statistically significant negative
value indicating sudden population growth (F S ¼ 4:86; p ¼ 0:041). However, when this
test was performed for the P. r. radula and P. r. oromii haplotypes separately, the
distribution for P. r. oromii was not statistically different than the one expected under a
scenario of constant population size (F S ¼ 0:65; p ¼ 0:62). A similar result was obtained
for P. canariensis (F S ¼ 1:09; p ¼ 0:08), although in this case the test can be
compromised due to the low sample size. Assuming a stepwise expansion model, the
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Fig. 3. Mismatch distributions for Pimelia COI DNA sequence subsets. The curves represent the observed relative
frequencies of nucleotide differences between pairs of individuals and the distribution fitted to the data under the
assumption of a model of population expansion. p-Value represents the probability that the variance of the
simulated data set is equal or greater than the observed one. (a) P. ascendens; (b) P. radula including the two
subspecies; (c) P. l. validipes; (d) P. l. laevigata (e) P. l. costipennis. Pimelia canariensis was not included in this
analysis due to the low number of individuals of the sample.
Table 2
Summary of parameters estimated assuming a stepwise population growth model in several Pimelia species from
the Canary Islands. The estimator of the expansion time (t) and the mutation parameters before (y0) and after (y1)
the expansions are given according to Schneider and Excoffier (1999). The formula t ¼ t=ð2uÞ was used to estimate
the time of population expansions taking a generation time of 1 year, where u corresponds to the mutation rate for
the DNA sequence used per generation. Confidence intervals for t are based on the maximum–minimum
nucleotide substitution rate values for arthropod mitochondrial DNA
Species
y0
y1
t
Time (Ma)
P.
P.
P.
P.
P.
4.116
0.004
0.509
0.451
0.000
42.461
7.525
60.439
19.441
3278.750
2.213
12.204
2.847
3.104
1.752
0.5370.04
2.9170.20
0.6870.05
0.7470.05
0.4270.03
ascendens
radula
l. validipes
l. laevigata
l. costipennis
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oldest population growth in Tenerife is estimated at about 2.970.2 Ma for P. radula. This
estimate is based on a time of the expansion t value of 12.204 and a mutation rate for the
considered sequence of u ¼ 2.15 106 per generation (see Material and Methods and
Table 2). Similarly, in P. ascendens the expansion could be dated at 0.670.04 Ma, while
P. laevigata expansions are estimated to be 0.6870.05 Ma for P. l. validipes (La Gomera)
and 0.7470.05 Ma for P. l. laevigata (La Palma) and 0.470.03 Ma for P. l. costipennis
(El Hierro).
3.4. Population history inferences from NCPA
The limit of mutational connections (probabilityX95% of being connected in a
parsimonious way) for our data set was five nucleotide substitutions using statistical
parsimony. The parsimony networks of COI haplotypes were resolved except for some
loops of ambiguity (haplotypes with more than one most parsimonious connection to the
rest of the network). We present in Fig. 4 the results for the preferred network, based on
geographical distribution of the mitochondrial genotypes and haplotype frequency criteria
(see Material and Methods). Four major subnetworks were obtained connected by more
than five steps. Haplotype group 5.1 includes all P. laevigata haplotypes and 5.2 contain P.
ascendens and P. canariensis haplotypes (equivalent to clade B1 in the tree of Fig. 2).
Group 5.3 contains P. r. radula and P. r. oromii haplotypes (corresponding to clade A2 in
the tree of Fig. 2) and 5.4 P. r. radula (Clade A1) exclusively. We used the maximum
parsimony tree (Fig. 2) to compute the steps connecting these higher categories; clades 5.1
and 5.2 are connected by nine steps, 5.3 and 5.4 by 10, and 6.1 and 6.2 are joined by 18
nucleotide substitutions.
At the total cladogram level, we explored two nesting alternatives. The more
conservative nesting option was considering the four 5-levels as unconnected (nesting I
in Fig. 4). The second option (nesting II in Fig. 4) was to build the last two nesting levels
connecting the clades 5.1–5.2 and 5.3–5.4 to obtain the higher 6.1 and 6.2 clades,
respectively, at the total cladogram level. These connections have a high probability of
being affected by homoplastic changes, but still this nesting design is a plausible
representation of the phylogenetic relationships if we take into account previous
phylogenetic information (Pons et al., 2004) and geographic-paleogeological considerations (i.e. distance between islands and their age of emergence; Juan et al., 1995; Juan et al.,
2000). In addition, distinguishing tip and interior clades at this nesting levels can be
ambiguous based on the tree of Fig. 2 or in the distance analysis, as the phylogenetic
relationships of clades A and B in are not well supported. For example, some haplotypes
(GM27) seem to be as close to the outgroup as any haplotype in clades A1 or A2. Instead,
we used a more robust Pimelia species-phylogeny using mitochondrial (COI+cytochrome
b, 16S RNA) and nuclear (28S RNA+Histone 3) sequences (Pons et al., 2004) to root at
clade 5.4 (P. r. radula) in nesting I option, while clade 6.2 was the root (interior) and 6.1 tip
in nesting II.
The association between genetic and geographic distribution was rejected at most low
nesting levels (1step clades) showing haplotypic variation. In the few cases where
significant associations were obtained, many gave inconclusive outcomes after following
the inference key of Templeton (2004) probably due to an inadequate sample design.
Contingency tests showed significant geographical associations of haplotypes contained
within some higher nesting levels, for example, 5.1 (P. laevigata), 5.2 (Tenerife clade B1)
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Fig. 4. Statistically preferred parsimony network and associated nesting design. The numbers included in circles
correspond to the haplotypes in each island (codes listed in Appendix A without the island identifying letters for
simplicity). The size of the circle is scaled to the number of individuals possessing that haplotype. Numbers with
an asterisk correspond to different haplotypes listed in Appendix A that are collapsed by the program TCS due to
nucleotide (nt) sequence size differences and consequently with missing data at particular nucleotide sites. Empty
circles indicate haplotype intermediates not present in the sample. Each connecting line represents a single
mutational step between any two given haplotypes. Dotted lines represent alternative ambiguous connections
(loops). The nested design is represented by increasing levels of nested boxes (see Material and Methods for
details). Clades for which significant association between genotypes and geography were obtained are labelled
with two numbers. The first one refers to the nesting level, and the second number identifies a particular clade at
this level (the missing ones gave non-significant results). A1, B, B1 and B2 relate to the particular clades in Fig. 2
and the island-specific haplotypes of P. laevigata are separated by three thick lines, the name of each island from
which the haplotypes come is indicated for clarity. The inset shows the two alternative nestings (I and II) at the
total cladogram level (see text for details). Connections by broken lines join major subnetworks separated by a
number of mutational steps higher than the limit of parsimony in alternative II. Two asterisks indicate root clades
deduced from the phylogeny obtained in Pons et al. (2004).
and 5.3 (Tenerife clade A2) (see Table 3). Using geographic distance analysis, the oldest
inferred event corresponding to the total cladogram was independent of the nesting design.
Both I and II nestings point to long distance colonizations from the north-east tip of
Tenerife to the west (see Fig. 5) although an alternative explanation of past fragmentation
followed by range expansion cannot be completely ruled out (see Discussion). The long
distance movement inference is obtained at other nesting levels as well for the clades within
6.1 (Nesting II), that is, Tenerife seems to have been the source of colonizers that
populated La Gomera. From the latter, subsequent distance dispersals colonized possibly
La Palma first (clade 5.1, inference of long distance colonization from La Gomera to La
Palma, see Fig. 5). This inference is reinforced by the result obtained at the 4.2 level,
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489
Table 3
Results of the nested cladistic phylogeographic analyses for Pimelia samples. Only instances of statistically
significant results obtained by geographical distance analysis are shown. The results at the one-step level are not
shown due to the low number of individuals (see Results). w2 statistics and the associated p-values were obtained
by permutational contingency tests with 10,000 replications. This test was non-significant only for clades 2.1 and
2.3, being the geographical distance analysis using Dc and Dn distances statistically significant
w2
Clade
Clades
2.1
Clades
2.3
Clades
2.9
Clades
2.12
Clades
2.15
Clades
3.4
Clades
3.5
Clades
3.10
Clades
4.2
p-Value
Inference chain
Inference
within
25.8678
0.2250
1–2–11–17-No
Inconclusive outcome
within
5.0000
0.4010
1–2–11–12-No
Contiguous range expansion
within
91.0350
0.0000
1–2–11–12-No
Contiguous range expansion
within
9.0000
0.0450
1–19–20–2–11–12-No
Contiguous range expansion
within
14.1429
0.0150
1–2–11–17-No
Inconclusive outcome
within
19.9500
0.0250
1–2–3–4-No
within
63.6849
0.0000
1–2–11–12-No
Restricted gene flow with
isolation by distance
Contiguous range expansion
within
18.0556
0.0270
1–2–3–4-No
within
127.9667
0.0000
1–2–3–5–6-Too few clades-7Yes
20.3667
0.0000
1–2–11–12-No
Restricted gene flow with
isolation by distance
Restricted gene flow/Dispersal
but with some long distance
dispersal
Contiguous range expansion
16.0000
0.0010
1–19–20–2–3–4–9-No
Allopatric fragmentation
121.0933
0.0000
1–2–3–5–15–21-Yes
Long distance colonisation
21.8169
0.0010
1–2–3–4-No
171.0000
0.0000
1–19–20–2–11–12–13–21-Yes
Restricted gene flow with
isolation by distance
Long distance colonisation
27.5550
0.0000
1–2–11–12-No
Contiguous range expansion
578.3400
0.0000
1–2–11–12–13–21-No
204.0000
0.0000
1–19–20–2–11–12–13–21-No
Long distance movements or
combined effects of gradual
movement during a past range
expansion and fragmentation
Long distance movements or
combined effects of gradual
movement during a past range
expansion and fragmentation
Clades within
4.3
Clades within
4.4
Clades within
5.1
Clades within
5.2
Clades within
6.1
Clades within
6.2
Total cladogram
I
Total cladogram
II
including haplotypes of P. laevigata from the three islands. Besides these results, two
instances of restricted gene flow with isolation by distance (clades 3.10 and 5.2) and one of
allopatric fragmentation (clade 4.4) are inferred within Tenerife. Most other nesting levels
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Fig. 5. Schematic representation of the Pimelia NCPA inferences overlaid on a map of the western Canary
Islands. Several instances of contiguous range expansion (CRE) are deduced within each island, and cases of
restricted gene flow with isolation by distance (RGF) in Tenerife and El Hierro. At least three long distance
colonizations (LDC) are inferred, represented in the figure showing east–west direction, although an alternative
explanation for the one at the total cladogram level is also possible (see Discussion). The origin of colonization for
El Hierro remains unclear. Numbers for each inference refer to nesting groups presented in Fig. 4 and Table 3.
with significant associations of genotypes with geography provide the general common
inference of contiguous range expansion in Tenerife (clades 6.2 north–north-east, and 4.3
in the central region), La Gomera (clades 3.5 and 2.12), La Palma (clade 2.3) and El Hierro
(clade 2.9). In the latter, a case of restricted gene flow with isolation by distance is deduced
(clade 3.4) between the south-west localities and the rest of the island. Clades 2.1 and 2.15
showed inconclusive results.
4. Discussion
In this study we have extended Juan et al.’s surveys (1995, 1996) of western Canary
Pimelia species by widening the sampling in Tenerife and collecting in the other three
western islands, where the related species P. laevigata occurs in unforested areas. If we take
into account geography, age of the islands and genetic data, P. laevigata populations in the
western islands seem to derive from a Tenerifean ancestor (Juan et al., 1995, 1996, this
work). The alternative hypothesis seems more unlikely as it would imply a backcolonization from these islands to Tenerife. Although the DNA fragment sequenced is too
short to provide enough phylogenetic information to support each P. laevigata island
population as unequivocally monophyletic, other genetic markers point to the
independence of the three clades (Pons et al., 2004).
The presence in La Gomera of two haplotypes (GM26 and GM27) that are relatively
divergent from the remaining haplotypes sampled on the island make it ambiguous to
postulate a single colonization event and/or estimate date(s) for them in this island. As
pointed out by Emerson et al. (2000c), the effect of existing genetic diversity in the
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491
ancestral population and the possibility of lineage extinction, complicate the inference
of colonization events from one island to another in a given phylogeny. In Tenerife,
the results obtained elsewhere (Juan et al., 1996) pointing out the existence of two
very divergent mitochondrial lineages are confirmed with our present more
comprehensive sampling. Lineage A includes P. radula haplotypes, with the current
subspecies designation not completely coincidental with lineages A1 and A2 since
haplotypes of P. r. radula are in both groups. This can be explained either by retention
of ancestral polymorphisms or, more probably, by mitochondrial introgression.
Nevertheless, after the analysis of the geographically intermediate, newly discovered
population in La Matanza (MT, Tenerife), we can state that the morphological characters
on which the two subspecies are based show a clinal variation along the north–north-east
region of Tenerife. On the other hand, Tenerife mitochondrial lineage B1 is the sister
group of P. laevigata, including P. ascendens distributed at relatively high altitude
localities, and P. canariensis present along the south-east to south-west coastal arid areas
of the island. In addition, the tree topology suggests that colonization to the western
islands (being probably La Gomera first to be colonized) was by ancestors of the B1
Tenerife lineage.
Pairwise differences between sampled DNA sequences (Fig. 3) show that in all the
studied cases, mitochondrial mismatch distributions are compatible with historic
population expansions or bottlenecks followed by a return to the original population size (Slatkin and Hudson, 1991; Rogers and Harpending, 1992). Estimates for
the corresponding dates of population expansion are in good accordance with the
island dates of formation (Table 2). For example, population expansion is inferred to
be older for the A lineage of Tenerife compared to one of the remaining species, while the
P. laevigata expansion in the El Hierro population is deduced to be more recent and
explosive (Table 2). This result is in accordance with the younger age for the subaerial
formation of this island and, therefore, within the time frame in which colonization was
possible.
One objective of this paper was to assess the validity of nested phylogeographical
cladistic analysis (Templeton et al., 1995; Templeton, 2004) in order to infer historical
population events in an a priori scenario of recurrent long distance dispersal, such as
the one assumed to explain colonization and diversification of an oceanic archipelago. The
Canary Islands are of independent volcanic origin and have never been connected by the
effect of sea level oscillations, with the possible exception of Fuerteventura and Lanzarote
that are separated by a very shallow strait, but whose populations are not included in this
study. As expected, molecular phylogenies show that in the Canaries the general pattern of
colonization in a diversity of terrestrial organisms is consistent with dispersal from older to
younger islands, westwards along the island chain (Juan et al., 2000). NCPA infers long
distance movements in cases of a large nesting distance (Dn) in a clade with small clade
distance (Dc) (though extinction of intermediate populations in organisms with low
vagility can also be compatible with this pattern). This inference is expected to occur, for
example, if a peripheral population has a unique haplotype, but a reduced geographical
distribution with respect to that of the haplotypes from which it derived, while, on the
contrary, concordant Dc and Dn values suggest short distance movements (Masta et al.,
2003). Assuming an adequate geographical sampling, NCPA should distinguish between
the above alternatives, but cases of misinterpretation have been shown to occur in
situations of gradual expansions or re-invasions of previous ranges (Masta et al., 2003). In
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the present data set, the NCPA analysis can detect at least three instances in which
geographical association of genotypes could be explained by long distance movements
of the species. Alternatively, the pattern could be explained by the combined effects
of gradual movement during a past range expansion and a subsequent fragmentation
(step 21 in the NCPA updated inference key, Templeton, 2004). The discrimination
between the two alternatives has shown to be only possible using outside independent
information (Masta et al., 2003; Templeton, 2004). Pimelia species are flightless, so passive
dispersal has to be invoked for these organisms, possibly through floating vegetation
drifted by the prevailing north-east–south-west sea currents in this part of the Atlantic.
In fact, the Canary archipelago has been colonized primarily by single or multiple
continental dispersal events in terrestrial organisms even in animals with low vagility
(Juan et al., 2000; Emerson, 2002). On the other hand, in a scenario of islands of
independent volcanic origin, the possibility of range expansions followed by fragmentation has to be in principle discarded to explain haplotype distribution patterns
among islands. Because of that, all the instances in which both alternative scenarios
were suggested by the inference key for clades including different islands were considered
as long distance movements. Nevertheless, colonization events of the western islands
of La Gomera, La Palma and El Hierro consistent with a minimum of two eastward
long distance dispersals, were recovered by the NCPA analysis, although the origin
of colonization for El Hierro remains unclear. Another inference frequently obtained
at other nesting levels (6.2, 4.3, 3.5, 2.12, 2.9 and 2.3) is contiguous range expansion,
deduced to have occurred more than once in Tenerife and in each of P. laevigata island
populations, presumably after the original colonizations. These inferences are consistent
with the deduced population growth by mismatch distribution analyses in the same clades
(Fig. 3). Finally, in Tenerife a particular case of allopatric fragmentation of the Izaña (IZ)
locality, and two instances of restricted gene flow with isolation by distance are deduced.
This can be related to the complex geological dynamics of the island, in which geographical
barriers and local extinctions due to volcanism are expected to have been particularly
frequent.
NCPA has indeed limitations inherent to the sampling scheme (size and number of sites)
and cases in which there is not enough resolution to detect past events (Templeton, 2004).
In addition, more serious limitations of the analysis occur when false inferences or
biological misidentifications are obtained. The original inference key (Templeton et al.,
1995) has been validated using biological examples for which strong prior evidence existed
for particular past events such as range expansion or fragmentation and updated to avoid
errors or ambiguities (Templeton, 1998, 2001, 2004). Cross-validation obtaining multilocus data from independently segregating DNA regions, is recommended for obtaining
robust inferences (Templeton, 2004). In our case, sampling size is scarce from some of the
geographical sites, and although the number of sampled localities is relatively high, a more
comprehensive sample scheme would be needed to cover the geographic micro-scale of the
high altitude regions in the western islands. These sampling limitations resulted in few
cases of significant geographical associations of haplotypes for which inconclusive
outcomes are obtained, although the low nesting level in which they occur is irrelevant for
the main conclusions of NCPA. Our results also show that there is enough variation in the
200 bp fragment and the pooling of data from a previous study allowed a comprehensive
NCPA. However, the relatively high divergence of mitochondrial lineages within Tenerife,
and the ones corresponding to island populations of P. laevigata, make the connections
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493
and designation of interior-tip status at the higher nesting level ambiguous and dependant
on external previous information. Better genetic resolution by longer mitochondrial DNA
sequences and independent genetic markers would clarify the above points further. Finally,
NCPA and the associated inference key present some difficulties when applied to
populations present in several volcanic islands as the ones studied here. Creer et al. (2001)
studied the phylogeography of bamboo viper in Taiwan and in the offshore Pacific, Orchid
and Green islands. Haplotypes of the two latter populations were omitted from the NCPA
analysis because the islands are of volcanic origin and never have been connected to
Taiwan and the study focused on historical terrestrial migrations. As mentioned above,
in the colonization and diversification of an oceanic archipelago, the a priori hypothesis
is long distance dispersal, but if the clades within a nesting clade are found in
separate islands (so in separate areas with no overlap by definition) the inference is
allopatric fragmentation. In addition, the inferences of long distance colonization with
subsequent fragmentation or past fragmentation followed by range expansion are
implicitly applied to historical terrestrial situations, being the application to oceanic
islands somewhat elusive.
In summary statistical phylogeography—and more specifically nested phylogeographic
cladistic analysis, provides an excellent opportunity to contrast current and historical
causes for the genetic and geographical distribution of darkling beetles in the Canary
Islands. There is a considerable genetic variation and geographical structure both in low
and high altitude arid region populations of the ancient, geologically complex island of
Tenerife. In contrast, the younger western islands show evidences of more recent
colonizations and subsequent range expansions of Pimelia populations derived from a
Tenerife ancestor, and, therefore, limited geographical structuring.
Acknowledgements
Giulia Paroni helped us with laboratory techniques. Heriberto López, Antonio J. Pérez,
Antonio Camacho, Rubén Barone and Jesús Alonso contributed collecting specimens. We
thank the suggestions made by Eduard Petitpierre and Jesús Gómez-Zurita. The comments
of David Posada and of an anonymous referee on a previous version of the manuscript
helped to improve the paper. This study is supported by the Spanish Ministerio de
Educación y Ciencia including European Union FEDER funds (project REN2003-00024).
The permits to collect beetles in the protected areas were obtained from the Viceconsejerı́a
de Medio Ambiente del Gobierno de Canarias and the corresponding Cabildos of the
islands, which occasionally provided accommodation.
Appendix A
Summary of Pimelia sampling in the western Canary Islands and in Tenerife. Species
localities and their codes, co-ordinates, haplotype distribution (number of individuals with
the same haplotype in parenthesis) and number of individuals collected at a given locality
are given. Total number of haplotypes is higher than the figures given in Table 1 because
the haplotypes with missing data were excluded from the analysis of nucleotide and
haplotype diversities. (Table A1)
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Table A1
Localities
Code
Co-ordinates
Haplotypes
N
Tenerife
Pimelia ascendens
Bco. Natero
Izaña
BN
IZ
16.841/28.284
16.500/28.305
1
11
Las Cañadas
Las Manchas
Pinar Chio
Puerto Erjos
San José de los Llanos
CD
MN
PC
PE
SJ
16.623/28.223
16.800v28.283
16.763/28.240
16.802/28.317
16.785/28.325
TF15
TF1, TF2(2), TF3, TF7, TF12(2),
TF19, TF20, TF21, TF23
TF1(2), TF2(5), TF3, TF13, TF18
TF14, TF17
TF3
TF16, TF25
TF8(2), TF9, TF10, TF11, TF22,
TF24
Pimelia canariensis
Armeñime
Faro de Rasca
Malpaı́s de Güı́mar
Mña. Roja
AM
FA
MG
MR
16.761/28.122
16.695/27.999
16.367/28.310
16.541/28.035
TF5, TF27
TF5
TF4
TF4, TF26(3)
2
1
1
4
Pimelia radula oromii
Las Teresitas
Las Lagunetas
TE
LG
16.185/28.507
16.409/28.413
6
8
Sta. Cruz
Los Rodeos
SC
RD
16.251/28.453
16.348/28.478
TF6(5), TF38
TF6, TF28, TF29(2), TF30, TF31,
TF35, TF36
TF6, TF39(2)
TF6(3), TF33, TF34, TF40
3
6
Pimelia radula radula
La Matanza
La Vera
MT
VE
16.460/28.442
16.536/28.400
TF32, TF37, TF41, TF42
TF43(5), TF44
4
6
La Gomera
Pimelia laevigata validipes
Cementerio Arure
CA
17.318/28.130
5
Lomo de la Peceña
El Apartadero
LP
AP
17.286/28.124
17.267/28.108
La Cruz de Marı́a
CR
17.256/28.112
Lomo de los Cardos
Torián
Pajarito
Majona
LO
TO
PA
MJ
17.249/28.098
17.241/28.107
17.233/28.108
17.122/28.128
GM3, GM8, GM18,GM25,
GM26
GM2, GM3(2), GM4(3), GM15
GM2(2), GM4, GM8, GM12,
GM21, GM22, GM23
GM2, GM14, GM16, GM17,
GM20, GM24
GM5(2), GM6(2), GM19, GM27
GM1(5), GM9, GM13
GM1, GM7(2)
GM1(3), GM10, GM11
La Palma
Pimelia laevigata laevigata
Los Llanos
El Remo
El Paso
LL
RM
EP
17.912/28.659
17.855/28.552
17.852/28.657
Fuencaliente
Refugio del Pilar
Casas de Taburiente
El Riachuelo
Puntagorda
FU
RP
CT
RI
PU
17.845/28.494
17.835/28.614
17.894/28.704
17.846/28.680
17.999/28.760
LP1(3), LP5(3), LP21
LP1, LP5(2), LP6
LP3, LP15, LP16, LP17, LP24,
LP25, LP26
LP3(2), LP7(2), LP13, LP14
LP1
LP1, LP20
LP1, LP4(2), LP18
LP1, LP11, LP22, LP23
10
2
1
2
7
7
8
6
6
7
3
5
7
4
7
6
1
2
4
4
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Table A1 (continued )
N
Localities
Code
Co-ordinates
Haplotypes
Roque de los Muchachos
Mazo
RO
MA
17.906/28.769
17.792/28.539
LP2, LP8, LP10, LP12
LP2, LP6, LP9, LP12, LP19
4
5
El Hierro
Pimelia laevigata costipennis
Frontera
La Caleta
Nisdafe
La Restinga
El Pinar
Jinama
Sabinosa
Montaña Caracol
Roque Salmor
FR
LC
NI
RE
PI
JI
SA
MC
RS
17.997/27.799
17.890/27.801
17.961/27.769
17.982/27.644
17.981/27.712
17.981/27.763
18.097/27.665
18.124/27.728
18.005/27.821
EH1, EH3
EH1, EH2, EH3, EH9
EH1(2), EH2(2)
EH2(2), EH8(3)
EH2(2), EH6(3), EH7(4)
EH2(4), EH12, EH14
EH4(3), EH5(2)
EH11, EH13
EH10
2
4
4
5
9
6
5
2
1
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