MOLECULAR
PHYLOGENETICS
AND
EVOLUTION
Molecular Phylogenetics and Evolution 31 (2004) 554–571
www.elsevier.com/locate/ympev
Phylogeny of a Macaronesian radiation: host-plant use
and possible cryptic speciation in Liparthrum bark beetles
Bjarte H. Jordal,a,b,* Lawrence R. Kirkendall,a and Kjetil Harkestada
a
Department of Zoology, University of Bergen, Allegt. 41, N-5007 Bergen, Norway
School of Biological Sciences, University of East Anglia, Norwich NR47TJ, UK
b
Received 25 March 2003; revised 15 September 2003
Abstract
The Macaronesian islands are well known for their unique endemic floras of woody plants. Many of these unusual plant groups
provide important novel resources for bark and wood boring beetles which breed in dead or moribund parts of their host plants. The
bark beetle genus Liparthrum exploits a wide range of unusual host plants and has its highest proportion of species living on the
Macaronesian Islands. We used DNA sequences of the mitochondrial Cytochrome Oxidase I gene and the nuclear Elongation
Factor 1a gene, and morphological characters, to estimate the phylogenetic relationships among species endemic to these archipelagos, and to trace the evolution of host-plant use. All parsimony and Bayesian analyses of the combined data, and maximum
likelihood analyses of the molecular data, showed that species associated with Euphorbia are monophyletic. We also found genetic
and subtle morphological evidence for three cases of cryptic speciation in one polyphyletic species associated with different
Euphorbia plants, showing that high levels of host specialisation can occur also in insects breeding in older and very dry, dead
plant tissues.
Ó 2003 Elsevier Inc. All rights reserved.
Keywords: Bayesian inference; Cape Verde; Canary islands; COI; EF-1a; Evolution: Euphorbia; Host plant use; Madeira; Maximum likelihood;
Parsimony
1. Introduction
The majority of herbivorous insects are associated
with only one or a few closely related host plants (Farrell et al., 1992; Futuyma and Mitter, 1996). Persistence
of host-plant use over evolutionary time scales is further
reflected by sister species that continue to use host plants
from the same plant genus or family after cladogenesis
(Mitter et al., 1991). Many of the best examples of such
phylogenetic constraints in host-plant use are taken
from herbivores feeding on live, healthy plants. In dying
or dead plants, on the other hand, most of the chemical
components are increasingly degraded during senescence, with exposure of internal tissues to air and to
enzymes from various decomposing organisms changing
the herbivore environment. Hence, we may expect fewer
*
Corresponding author.
E-mail address: [email protected] (B.H. Jordal).
1055-7903/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved.
doi:10.1016/j.ympev.2003.09.008
dietary constraints on late-stage herbivores, resulting in
more frequent shifts to unrelated host-plant families.
In bark beetles feeding and breeding under bark of
dead trees, most species nevertheless show preference for
only a few congeneric tree species (Deyrup and Atkinson,
1987; Noguera-Martinez and Atkinson, 1990) and recent
phylogenetic studies have shown that these insects may
have long histories of host plant fidelity (Kelley and
Farrell, 1998; Sequeira et al., 2000). Bark-boring beetles
as a whole are more specialised to their host plants than
are, for instance, wood- or twig-boring beetles, or funguscultivating beetles which live in a mutualistic association
with their fungal symbionts in wood (Beaver, 1979). Although apparent host specificity in bark beetles could
result from the convenience of local host abundance rather than host constraints per se (see, e.g., Stevens, 1986),
evidence to the contrary has been found in at least one
genus of bark beetles (Kelley and Farrell, 1998).
Our study explores the evolution of host-plant use
in Liparthrum bark beetles found in very different
B.H. Jordal et al. / Molecular Phylogenetics and Evolution 31 (2004) 554–571
environments ranging from dry scrublands to more
humid forests, with particular focus on the euphorbiaceous scrublands and mountainous laurel forests of the
Macaronesian islands. The floras of Cape Verde, Canary
Islands, and Madeira have high proportions of endemic
woody plants (e.g., Barber et al., 2002) , many of which
are unique host plants for bark beetles (Wood and
Bright, 1992) and hence provide a special opportunity to
explore evolution of bark beetle host-plant use. Several
of the known host plants for Liparthrum are not only
very abundant throughout many of the islands, but their
distributions largely overlap. Preference for one species
of host plant then may reflect specialisation rather than
local adaptation to the most abundant (and suitable)
plant species. Among the 17 described species of
Liparthrum in Europe and Northern Africa, 12 are
found on the Macaronesian islands, 11 of which are
endemic. Compared to the total of 38 currently described species scattered throughout the mostly warm
and dry parts of the globe (Wood and Bright, 1992),
these islands contain the highest proportion of Liparthrum species for any area of comparable size. Apparent
adaptation to warm and dry climates is reflected in the
ability of a majority of species to breed in very dry wood.
Other aspects of their biology are more similar to typical
bark beetle behaviour, with monogamous pairs mating
in cave-like niches under the bark of dead trees and
shrubs, and larvae feeding upon phloem and wood while
tunnelling away from the parental cave (Israelson, 1990).
Phylogenetic information plays a crucial role in determining the importance of historical host fidelity versus
the rate and direction of host switching in herbivores. We
here estimate phylogenetic relationships among all but
one of the Macaronesian species and test their monophyly
using continental species of Liparthrum, the putative sister genus Hypoborus, and several other outgroup taxa.
We further ask if groups of species associated with the
same plant genus or family are monophyletic, suggesting
phylogenetic constraints on host plant use. In particular
we seek to elucidate the association with several arborescent species of the abundant Euphorbia spurges, a
group of plants well known for their wide range of toxic
compounds (Seigler, 1994). Previous taxonomic treatments have considered all Liparthrum species breeding in
Euphorbia to be a monophyletic group (Israelson, 1990;
Schedl et al., 1959; Wollaston, 1862) , which then provides
a logical null hypothesis to test. We also use a subsample
of beetles feeding on this plant genus to trace host specificity at the population level, thus testing a second hypothesis that populations feeding on different species of
Euphorbia are genetically distinct. Because Liparthrum
beetles breed in very old and dry woody material, the
most logical null hypothesis may be one of no association
with a particular group of euphorb host plants.
We used the mitochondrial gene Cytochrome Oxidase
I, the nuclear gene Elongation Factor-1a, and mor-
555
phological characters to reconstruct the phylogenetic
history of host-plant use and associated genetic variation. Molecular data are necessary to resolve the phylogeny of this group because morphological differences
between species are few and often ambiguous (Israelson,
1990). Such morphological uniformity may suggest that
Liparthrum as a whole, and the Macaronesian species in
particular, are recently derived, and have had little time
for morphological differentiation. On the other hand,
the scattered world-wide distribution of the genus has
led some authors (e.g., Wood, 1986) to argue that these
are relict species, remnants of a much more evenly distributed ancient clade of species. If so, we expect deep
divergences between many of the lineages, possibly
confusing phylogenetic signal at the early stages of diversification. A molecular phylogenetic analysis of Liparthrum will enable the evaluation of the possible relict
nature of the group.
2. Materials and methods
2.1. The Macaronesian islands
Macaronesia includes four Atlantic archipelagos west
of the African coast: the Azores, Madeira, Canary, and
Cape Verde island groups. No Euphorbia associated
beetle species are found on the Azores, and only one
species of Euphorbia and Liparthrum are found on the
Cape Verde archipelago (west of Senegal). The remaining species are found on the Madeiran islands (including
Porto Santo) and Canary Islands (El Hierro, La Palma,
La Gomera, Tenerife, Gran Canaria, Fuerteventura,
Lanzarote, and north of these, the Salvagem Islands).The
oldest islands in each archipelago are those which are
closest to the mainland (Fuerteventura, Porto Santo, and
the eastern Cape Verde islands) and these are known to be
at least 18 My, with younger islands gradually decreasing
in age westward, with the most recent island (El Hierro)
being only 1.1 My (summarised in Brown et al., 2001;
Hess et al., 2000).
2.2. Host plants
Records of host-plants were assembled from the literature (Wood and Bright, 1992) , and new records were
added after extensive recent field work. Castanea sativa
is a new record for both L. curtum and L. Ôrumicis,Õ and
the association of the latter species with Polygonaceae
(Rumex lanarius) is the first documented host-plant record from this plant family. Up to seven Liparthrum
species (depending on the status of undescribed species)
breed in only one of two different groups of Euphorbia
plants. The two groups are distinguished by toxic
(Euphorbia lamarckii group) vs. non-toxic (Euphorbia
balsamifera) latex, and by morphological and deep
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B.H. Jordal et al. / Molecular Phylogenetics and Evolution 31 (2004) 554–571
molecular divergence among the two groups. The species in the E. lamarckii group (Euphorbia regis-jubae,
Euphorbia piscatorium, Euphorbia anachoreta, Euphorbia
tuckeyana, E. lamarckii) are difficult to distinguish, have
very similar chemical composition, and are almost
identical at the nuclear ITS gene (Molero et al., 2002).
Hence we treated the latter group as a single functional
host plant.
2.3. Beetle samples
Most species of Liparthrum outside Macaronesia and
the Mediterranean area are rarely collected and have
only been found once or a few times, limiting the
number of available outgroups within Liparthrum.
Further outgroup species were therefore selected from
Hypoborus (monotypic) and Chaetophloeus in the same
tribe (Hypoborini), as well as a wider range of putatively
close outgroup species in other tribes (see Farrell et al.,
2001). Details about the specimens collected and sequenced, their geographical sources and host plants, are
listed in Table 1. Vouchers are in the senior authorÕs
collection and consist of siblings stored in 96% ethanol.
Multiple specimens per species were sequenced when
available from different localities, to assess intraspecific
variation and to test for species monophyly. Three undescribed species were given the informal names
L. ÔpilosumÕ, L. Ôrumicis,Õ and L. Ôpinicola,Õ corresponding to descriptions in progress (Knizek, in prep). Species
were defined primarily on the basis of distinct morphological differences. Morphologically and ecologically
similar populations which were genetically deeply
divergent from each other were subject to further
scrutiny of species status. Thus, the references to L.
ÔpilosumÕ A, B, and C were posterior assessments based
on the observation of deep coalescence among the three
populations.
2.4. Morphological characters
Most morphological variation in Liparthrum is difficult to divide into discrete characters. While many species can be readily identified by unique combinations of
characters, most of these are autapomorphies and only a
few are shared with other species. Thus only five clearly
separable and informative characters were included in
the combined phylogenetic analyses (see Appendix A).
2.5. DNA amplification and sequencing
DNA was extracted from single individuals with the
Qiagen DNeasy kit and partial gene fragments of Cytochrome Oxidase 1 (COI) and Elongation Factor 1-a
(EF-1a) were amplified using primers described by
Normark et al. (1999). Most PCR reactions were performed in a 25 ll volume containing 0.2 lM of each
primer, 0.2 mM of each dNTP, 0.5 U of Bioline BioTaq
DNA polymerase, 1 buffer with MgCl2 to a final
concentration of 1.5–2.0 mM. Typical PCR cycles (COI)
consisted of 90 s initial denaturing at 94 °C, followed by
38 cycles of 94 °C for 30 s, 46 °C annealing for 60 s, and
72 °C extension for 60 s, followed by a final extension for
7 min. For EF-1a we used a touchdown profile consisting of 43 cycles where all cycles had 72 °C extension for
40 s and 94 °C denaturing for 30 s, with the first 16 cycles
having decreasing annealing temperature from 60 to
47 °C by 2 °C (last 1 °C) every second cycle, with the
final 27 cycles at 46 °C for 60 s. Purified PCR products
were sequenced using a standard protocol for big-dye
version 2 (Perkin–Elmer).
Sequences were assembled and edited with Lasergene
software (DNASTAR). There are two copies of EF-1a
known from beetles (Jordal, 2002), and we used the
single-intron copy (C1) in this study. For COI and the
EF-1a coding region, alignments were unambiguous due
to the lack of any insertions or deletions in the coding
region. The EF-1a intron was aligned with the ClustalX
software (Thompson et al., 1997) and included in all
phylogenetic analyses. We performed alignments under
a wide range of gap opening and gap extension costs,
with transversions weighted equally or twice the transitions. Maximum parsimony (MP) searches were performed on each of the ClustalX alignments, with gaps
treated as a fifth character. Three of the alignments were
equally parsimonious, and one of these was selected
based on the highest number of shared nodes with the
exon tree (gap cost ¼ 3, extension cost ¼ 1.5, and tv ¼ 2).
Sequences are deposited in GenBank under the accession numbers (COI) AY376998–AY377061 and (EF-1aÞ
AY377062–AY377123.
2.6. Phylogenetic analyses
We used Paup* 4.0 (Swofford, 2002) to calculate
pairwise sequence divergences, base composition, tree
statistics, and phylogenetic analyses under the parsimony and likelihood criteria. MacClade 3.04 (Maddison
and Maddison, 1992) was used for editing matrices and
calculating substitution frequencies. Under the maximum parsimony criterion, we performed 200 random
addition replicates of heuristic searches for each of the
data partitions and the combined matrix. Bootstrap
support (BP) for individual nodes (Felsenstein, 1985)
was assessed by 200 bootstrap replicates of 10 random
addition searches. TreeRot v2 (Sorenson, 1999) was
used to calculate Bremer support (BS: Bremer, 1994)
and partitioned Bremer support indices (PBS: Baker and
DeSalle, 1997) showing the relative contribution from
each data partition to the combined analysis. We used
default settings during the heuristic Bremer support
searches, with 20 random addition sequences per constrained node. Although we follow the convention of
Table 1
Details on collection localities, host plants, and species distribution for the species included in this study
Species
Locality
Collector
Host plant
Greece, Naxos, 1.7.1999
Madeira, Machico, 1.5.2000
Italy, Brescia, 23.8.2001
Morocco, TiziÕn Test, 21.4.2002
L. Kirkendall
K. Harkestad
M. Faccoli
B. Jordal
Ficus
Ficus
Ficus
Ficus
Liparthrum artemisiae
Liparthrum artemisiae
CI, La Gomera, Tamargada, 15.8.1999
CI, Tenerife, Buenavista, 17.8.1999
K. Harkestad
K. Harkestad
Artemisia canariensis
Artemisia canariensis
Liparthrum bartschti *
Austria, Vienna, 1.3.1992
M. Knizek
Viscum album
Liparthrum
Liparthrum
Liparthrum
Liparthrum
CI,
CI,
CI,
CI,
K. Harkestad
B. Jordal
K. Harkestad
K. Harkestad
Euphorbia
Euphorbia
Euphorbia
Euphorbia
Hypoborus
Hypoborus
Hypoborus
Hypoborus
ficus
ficus
ficus *
ficus *
La Gomera, Bco. Santiago, 4.8.1999
La Gomera, Playa de la Caleta, 9.3.2002
Tenerife, Buenavista, 17.8.1999
Tenerife, El Escobonal, 12.8.1999
balsamifera
balsamifera
balsamifera
balsamifera
Liparthrum bituberculatum
Liparthrum bituberculatum
Liparthrum bituberculatum
CI, La Gomera, Epina, 15.8.1999
Madeira, Ribeiro Frio, 5.5.2000
CI, Tenerife, Tunel Bailadero, 20.8.1999
K. Harkestad
K. Harkestad
K. Harkestad
Laurus azorica
Laurus azorica
Laurus azorica
Liparthrum canum
CI, La Gomera, Archejo, 15.8.1999
K. Harkestad
Echium aculeatum
Liparthrum colchicum *
Liparthrum colchicum *
Spain, 1998
Spain, 1998
M. Lombardero
M. Lombardero
Laurus nobilis
Laurus nobilis
Liparthrum
Liparthrum
Liparthrum
Liparthrum
Liparthrum
curtum
curtum
curtum
curtum
curtum
CI, La Gomera, Agulo, 13.8.1999
Madeira, Camara de Lobos, 30.4.2000
Madeira, Curral das Freiras, 2.5.2000
CI, Tenerife, Arona, 19.8.2002
CI, Tenerife, Taimano, 11.8.2003
K.
K.
K.
K.
K.
Ficus carica
Ficus carica
Castanea sativa
Ficus carica
Ficus carica
Liparthrum
Liparthrum
Liparthrum
Liparthrum
inarmatum
inarmatum
inarmatum
inarmatum *
Madeira, Port Santo, 7.9.2002
Madeira, Porto Moniz, 3.5.2000
Madeira, Ribeira Brava, 5.9.2002
CI, Tenerife, Punta de Hidalgo, 16.2.2002
B. Jordal
K. Harkestad
B. Jordal
B. Jordal
Euphorbia
Euphorbia
Euphorbia
Euphorbia
piscatoria
piscatoria
piscatoria
lamarckii
Liparthrum
Liparthrum
Liparthrum
Liparthrum
loweanum
loweanum
loweanum
loweanum
CV,
CV,
CV,
CV,
B.
B.
B.
B.
Jordal
Jordal
Jordal
Jordal
Euphorbia
Euphorbia
Euphorbia
Euphorbia
tuckeyana
tuckeyana
tuckeyana
tuckeyana
Liparthrum
Liparthrum
Liparthrum
Liparthrum
lowei
lowei
lowei
lowei
CI,
CI,
CI,
CI,
K.
K.
K.
K.
Harkestad
Harkestad
Harkestad
Harkestad
Euphorbia
Euphorbia
Euphorbia
Euphorbia
lamarckii
lamarckii
lamarckii
lamarckii
Santo Antao, Fontheinas, 8.10.2002
Santo Antao, Rabo Curto, 9.10.2002
Sao Vicente, Monte Verde, 7.10.2002
Sao Vicente, Monte Verde, 7.10.2002
La Gomera, Hermigua, 13.8.1999
La Gomera, Hermigua, 15.8.1999
Tenerife, Bajamar, 18.8.1999
Tenerife, La Orotava, 18.8.1999
Harkestad
Harkestad
Harkestad
Harkestad
Harkestad
M
S
H
P
G
T
C
F
L
A
E
CI, La Gomera, Las Hayas, 14.8.1999
CI, Tenerife, Santiago Teide, 22.8.1999
CI, Gran Canaria, Las Lagunetas, 22.2.2002
K. Harkestad
K. Harkestad
B. Jordal
Cytisus proliferus
Cytisus proliferus
Laurus azorica
Liparthrum Ôpilosum-AÕ-1
Liparthrum Ôpilosum-AÕ-2
CI, Gran Canaria, Galdar, 3 km S, 22.2.2002
CI, Gran Canaria, Agaete, 8 km S, 20.2.2002
B. Jordal
B. Jordal
Euphorbia regis-jubae
Euphorbia regis-jubae
557
Liparthrum nigrescens
Liparthrum nigrescens
Liparthrum nigrescens *
B.H. Jordal et al. / Molecular Phylogenetics and Evolution 31 (2004) 554–571
bicaudatum
bicaudatum
bicaudatum
bicaudatum
V
carica
carica
carica
carica
558
Table 1 (continued)
Locality
Collector
Host plant
Liparthrum Ôpilosum-AÕ-3
CI, Gran Canaria, Galdar, 3 km S, 22.2.2002
B. Jordal
Euphorbia regis-jubae
Liparthrum
Liparthrum
Liparthrum
Liparthrum
Liparthrum
Liparthrum
Liparthrum
Ôpilosum-BÕ
Ôpilosum-BÕ
Ôpilosum-BÕ
Ôpilosum-BÕ
Ôpilosum-BÕ-1
Ôpilosum-BÕ-2
Ôpilosum-BÕ-3
CI,
CI,
CI,
CI,
CI,
CI,
CI,
B. Jordal
B. Jordal
K. Harkestad
B. Jordal
B. Jordal
B. Jordal
K. Harkestad
Euphorbia
Euphorbia
Euphorbia
Euphorbia
Euphorbia
Euphorbia
Euphorbia
balsamifera
balsamifera
balsamifera
balsamifera
balsamifera
balsamifera
sp.
Liparthrum
Liparthrum
Liparthrum
Liparthrum
Ôpilosum-CÕ
Ôpilosum-CÕ
Ôpilosum-CÕ
Ôpilosum-CÕ
CI, La Gomera, El Cercado, 10.3.2002
CI, La Palma, Puntallana, 28.2.2002
Salvagem Islands, 25.5.1999
Salvagem Islands, 25.5.1999
B. Jordal
B. Jordal
M. Arechevaleta
M. Arechevaleta
Euphorbia
Euphorbia
Euphorbia
Euphorbia
lamarckii
lamarckii
anachoreta
anachoreta
Liparthrum
Liparthrum
Liparthrum
Liparthrum
ÔpinicolaÕ
ÔpinicolaÕ
ÔpinicolaÕ
ÔpinicolaÕ
CI,
CI,
CI,
CI,
B. Jordal
B. Jordal
K. Harkestad
K. Harkestad
Pinus
Pinus
Pinus
Pinus
El Hierro, Charco Manso, 6.3.2002
Fuerteventura, Pajara, 24.2.2002
La Gomera, Bco. Santiago, 14.8.1999
La Palma, Las Indias, 3.3.2002
Tenerife, Roque de las Bodegas, 14.2.2002
Tenerife, Los Cristianos, 16.2.2002
Tenerife, Buenavista, 17.8.1999
El Hierro, Las Casillas, 7.3.2002
La Palma, Monte de Luna, 3.3.2002
Tenerife, Arona, 19.8.1999
Tenerife, Arona, 19.8.1999
V
S
H
P
G
T
C
F
L
A
E
canariense
canariense
canariense
canariense
Liparthrum ÔrumicisÕ
Liparthrum ÔrumicisÕ
Liparthrum ÔrumicisÕ
CI, El Hierro, Sabinosa, 6.3.2002
CI, Tenerife, Arona, 19.8.1999
CI, Tenerife, Arona, 19.8.1999
B. Jordal
K. Harkestad
K. Harkestad
Rumex lunarius
Castanea sativa
Castanea sativa
Liparthrum semidegener
Madeira, Ribeira Frio, 4.9.2002
B. Jordal
Teline madeirense
Outgroups:
Chaetophloeus penicillatus
Acacicis sp.
Ficicis despectus
Pseudochramesus sp.
Ctonoxylon flavescens
USA, Arizona: Chiricahua, 28.8.1998
Australia, Queensland, Conondale, 20.1.2000
Papua New Guinea, Madang, 29.1.2000
Argentina, Salta, Enrique Moscone, 18.2.1998
Uganda, Budongo Forest, 3.7.1998
B.
B.
B.
B.
B.
Rhus trilobata
Unknown
Ficus sp.
Unknown
Ficus sp.
Jordal
Jordal
Jordal
Jordal
Jordal
M
Individuals marked by a Ô*Õ were pruned from the matrix during ML analyses, due to long branches (L. bartschti) or >30% missing data in one or both of the gene partitions. CI ¼ Canary Islands,
CV ¼ Cape Verde Islands. Islands and continents are indicated as follows (in italics, the seven Canary Islands): V, Cape Verde Islands; M, Madeira islands; S, Salvagem Islands; H, El Hierro; P , La
Palma; G, La Gomera; T , Tenerife; C, Gran Canaria; F , Fuerteventura; L, Lanzarote; A, Africa (Morocco, Algerie); E, Europe (mostly Medirerranean area). The designations of ÔpilosumÕ A, B, and
C, are posterior assessment based on deep genetic divergence at nuclear and mitochondrial loci.
B.H. Jordal et al. / Molecular Phylogenetics and Evolution 31 (2004) 554–571
Species
B.H. Jordal et al. / Molecular Phylogenetics and Evolution 31 (2004) 554–571
559
We applied a one-tailed test of significance and asked
whether our observed number of steps was significantly
lower than the number of steps observed on the random
topologies. Given that E. balsamifera and species of the
E. lamarckii group completely overlap throughout the
Canary Islands but not in Madeira or the Cape Verde
Islands, we only tested individuals collected on the former islands.
always combining data to maximise explanatory power
(Kluge, 1989), we also examined properties of each data
set to infer sources of incongruence and differential
contribution to the total evidence results. We further
explored nodes that were incongruent across partitions
and optimality criteria, applying a variety of weighting
schemes for the EF-1a intron, COI third positions, and
morphological characters.
Parameters for maximum likelihood (ML) analyses
were estimated with Modeltest 3.06 (Posada and Crandall, 1998) which uses the neighbour joining algorithm
via Paup*. ML searches were performed on a data
matrix where we excluded five taxa which contained
substantial amounts of missing data (>30%) in one or
both partitions. MrBayes 3.0b4 (Huelsenbeck and
Ronquist, 2001) was used to calculate posterior probabilities under the likelihood criterion. For each analysis,
we used the default settings of three heated and one cold
Markov chains run in parallel with swapping between
chains for 500,000 generations. Trees were sampled every 500 generations with the first 300 trees (150,000
generations) discarded as burn-in (likelihoods below
stationarity level). We also estimated 95% credibility
intervals for all parameters, to assess levels of parameter
overlap in the two gene partitions (Buckley et al., 2002).
Bayesian analyses on the combined data were performed
with a mixed model, estimating model parameters separately for each data partition (COI, EF-1a, and morphology), and compared to a single model for all
partitions to explore the effect of combining differently
evolving partitions.
We tested the hypothesis that the observed association with a single group of Euphorbia (balsamifera or
lamarckii group) was significantly different from an arbitrary pattern (resulting from limited field collections)
by mapping host-plants on 1000 random tree topologies.
3. Results
3.1. Outgroup selection
A preliminary parsimony analysis of the combined
data from all partitions showed that Hypoborus and
Liparthrum are monophyletic with respect to all outgroups tested. Liparthrum bartschti was the sister group
to all remaining Liparthrum, including Hypoborus
(bootstrap support, 90; Bremer support, 7). The node
separating L. bartschti and Hypoborus from the remaining Liparthrum was also well supported (bootstrap
support, 99; Bremer support, 9). All outgroup taxa, including Chaetophloeus, were highly divergent from the
ingroup as indicated by a Bremer support value of 111
for the node leading to the ingroup. Hence, L. bartschti
and Hypoborus ficus were used as functional outgroups
in all further analyses to avoid randomization of character polarity and spurious parameter estimation.
3.2. Genetic variation
Uncorrected intraspecific variation in the COI gene
ranged between 0.6% in L. bituberculatum and 10.4% in
L. loweanum, with six species exceeding 5% (Table 2).
This variation was much lower in EF-1a than in COI, in
Table 2
Comparison of maximum intra-specific sequence variation (uncorrected p-distances) for species collected from two or more islands
Species
Host plant range
N ind.
H. ficus
L. artemisiae
L. bicaudatum
L. bituberculatum
L. curtum
L. loweanum
L. lowei
L. nigrescens
L. inarmatum
L. Ôpilosum-BÕ
L. Ôpilosum-CÕ
L. ÔpinicolaÕ
L. ÔrumicisÕ
L. inarmatum-complex
Ficus carica
Artemisia canariense
Euphorbia balsamifera
Laurus azorica
F. carica, Castanea sativa
E. tuckeyana
E. lamarckii
L. azorica, Cyticus proliferus
E. lamarckii, E. piscatorium
E. balsamifera
E. lamarckii, E. anachoreta
Pinus canariense
Castanea sp, Rumex lunarius
Euphorbia spp.
4
2
4
3
5
4
4
3
4
6
4
4
3
18
4
2
2
3
3
2
2
3
3
5
3
3
2
9
6.7
5.0
2.4
0.6
9.5
10.4
3.6
8.5
1.5
1.6
4.6
1.8
1.2
14.0
1.4
1.3
0.6
0.2
0.4
1.6
0.7
1.2
0.1
0.4
1.3
2.1
0.6
2.1
2.6
2.6
0.0
0.0
2.6
5.3
2.6
13.2
2.6
10.5
15.8
5.3
2.6
18.4
Macaronesian Lip.
All Liparthrum
cf. Table 1
cf. Table 1
54
55
11
11+
18.4
23.7
7.9
13.9
31.6
31.6
The range of host plants for the sequenced beetles is also included.
N isl.
CO1
EF exon
EF intron
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B.H. Jordal et al. / Molecular Phylogenetics and Evolution 31 (2004) 554–571
particular for the exon fragment, and ranged between
0.2 and 2.1% for the same species. The correlation of
sequence divergence between the two genes was not very
strong, apparently due to most interspecific comparisons
exceeding 10% (uncorrected) in COI, approaching
saturation. Saturation in COI was evident from plotting
sequence divergences for both genes, corrected for
multiple hits using models of evolution estimated by
Modeltest (Fig. 1A). The intron showed a rather unpredictable pattern of variation (Table 2), but intron
sequence divergence correlated well with that of the
exon (Fig. 1B) and was included in all further analyses
of EF-1a (as recommended in previous work: Jordal,
2002; Kawakita et al., 2003).
The higher level of saturation in COI was also reflected in a higher level of homoplasy, measured by
substitutional changes over the topology resulting from
an unweighted MP search (Fig. 2). Both intron and exon
(mostly third positions) of the EF-1a had distinctly
more left-skewed distributions of substitutional changes
compared with COI third positions. The latter sub
partition was also the only one with a significant bias in
base composition (v2 ¼ 211:55, P ¼ 0:008).
3.3. Separate phylogenetic analyses
The COI alignment for Liparthrum and Hypoborus
consisted of 675 base pairs, 239 of which where parsimony
informative. Heuristic parsimony searches with 59 sequences resulted in 288 MP trees; the strict consensus tree
is presented in Fig. 3. The functional ingroup of primarily
Macaronesian species was highly supported (bootstrap
support ¼ 93) whereas most other basal nodes were not
supported. Most species were monophyletic and well
Fig. 1. Plots of pairwise sequence comparisons for the 52 taxa used in all analyses. (A) COI vs. EF-1a ML-corrected distances; (B) EF-1a intron vs.
EF-1a exon p-distances. Maximum likelihood parameters used to calculate ML distances are given in Table 4.
Fig. 2. Histograms showing the number of substitutions required for each character, measured for each gene over the total evidence MP topology
(Fig. 5).
B.H. Jordal et al. / Molecular Phylogenetics and Evolution 31 (2004) 554–571
561
Fig. 3. Strict consensus trees resulting from the separate parsimony analyses of the two gene partitions. CO1, 488 trees of length 1279 steps, CI ¼ 0.32
(informative only), RI ¼ 0.76; EF-1a, 60 trees of length 601 steps, CI ¼ 0.53, RI ¼ 0.82. Deletion of the additional taxa which lacked EF-1a sequences
did not affect the CO1 topology (includes L. colchicum and L. inarmatum-T). Arrows point to nodes defining single origins of feeding behaviour.
Acronyms for the geographic origin of samples are shown by: Madeira, PS, Porto Santo (small island off the coast of Madeira); Canary Islands, EH,
El Hierro; LP, La Palma; LG, La Gomera; T, Tenerife; GC, Gran Canaria; F, Fuerteventura; Cape Verde Islands (CV), SA, Santo Antao; SV, Sao
Vicente.
supported, except for the paraphyletic L. loweanum (with
respect to L. ÔpilosumÕ). The Laurus associated L. nigrescens, L. bituberculatum, and the continental L. colchicum
were monophyletic with strong BP support. Another well
supported multi-species clade consisted of L. semidegener,
L. canum, and L. artemisiae.
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B.H. Jordal et al. / Molecular Phylogenetics and Evolution 31 (2004) 554–571
The COI topology estimated by the ML and Bayesian
criteria differed from the MP trees in several ways
(Fig. 4). L. nigrescens was the sister species to L. Ôrumicis,Õ splitting the latterÕs relationship to L. Ôpinicola.Õ
Second, L. lowei was moved from its basal position to a
sister relationship with L. curtum. All changes were
characterised by very short internodes, with low posterior probabilities, further emphasising the lack of node
support in the MP analysis. Noteworthy instances of
long intraspecific branches was observed in the paraphyletic Cape Verdian L. loweanum, and in the monophyletic L. nigrescens, L. curtum, and L. artemisiae.
EF-1a consisted of 828 coding characters (157 informative) and one aligned intron of 73 characters, including gaps (49 informative). All species were
monophyletic in the parsimony analysis except for two
sequences of L. Ôpilosum-BÕ which grouped with
L. loweanum and L. Ôpilosum-AÕ (Fig. 3). Again the
functional ingroup was well supported, but several other
basal nodes were also supported, albeit modestly. The
well supported clade consisting of L. ÔrumicisÕ and
L. ÔpinicolaÕ was placed in a basal position among the
Macaronesian species. Another well supported clade,
artemisiae–canum–semidegener, was placed with medium
support as the sister group to all euphorb associated
species (L. lowei, L. loweanum, L. Ôpilosum,Õ L. bicaudatum, and L. inarmatum).
The ML and Bayesian analyses of EF-1a data resulted in a topology somewhat similar to the MP analysis, but differed by the basal placement of L. nigrescens,
and the paraphyly of L. Ôpilosum-CÕ with respect to
L. inarmatum (Fig. 4). All branch lengths were less then
five times as long as those in the COI topology, but the
ratio between specific (species) nodes and basal interspecific nodes were similar to COI. All basal nodes
contained low posterior probabilities with most nodes
leading to intraspecific clusters had maximum posterior
probabilities.
3.4. Combined analyses
The combination of all taxa and all data in the unweighted parsimony analysis (including five morphological characters, see Appendix A), resulted in 24 MP
trees. The strict consensus of these trees was identical
when taxa with missing data were excluded, and differed
from the initial outgroup analysis only by the polytomy
within the Euphorbia associated clade (Fig. 5). This topology only contradicts the results from the separate
EF-1a analysis by the clustering of L. bituberculatum
and L. colchicum with L. nigrescens, and with these three
species being sister group to the artemisiae–canum–
semidegener clade. However, when the highly saturated
COI partition was down weighted two, five, or 10 times
(entire gene or only third positions, see Table 3), the
sister relationship among these two clades disappeared.
The new sister relationship between the artemisiae clade
and the Euphorbia associates, and the monophyly of all
Euphorbia associated species, were increasingly supported under the same weighting scheme.
The three partitions contributed differential support
to the various hierarchical levels in the parsimony phylogeny. Although COI contributed the largest overall
Bremer support, most of the support was allocated in
terminal clades, contributing 10 times more support
than EF-1a at intraspecific levels (see Fig. 5). This difference was much less at deeper phylogenetic nodes. The
five morphological characters did not much influence
the overall topology, but the monophyly of the euphorb
associated species in the unweighted parsimony analysis
was dependent on two synapomorphic characters (Appendix A: characters 1, 3). However, the importance of
morphological characters disappeared when COI third
positions were down weighted five times or more (see
Table 3).
ML parameters for the combined gene partitions
were estimated and compared to the separate estimates
for each partition. We further estimated the 95% posterior intervals of these parameters, using empirical base
frequencies as known priors (Table 4). The shape of the
gamma distribution, the proportion of invariant sites,
and five of the substitution types were widely overlapping among the two gene partitions. Major discrepancies between the two models were thus confined to the
different proportions of guanine bases and rates of adenine–guanine substitutions. Hence we applied a mixed
model in the Bayesian analysis of the combined data
(including morphological data) and compared the resulting topology to the best single-model topology from
likelihood and Bayesian analyses. Bayesian analyses
under a single or mixed model produced identical topologies (Fig. 6), suggesting that a mixed model is not
always superior to a single model. The likelihood topology differed from the Bayesian topology primarily by
the more basal position of L. nigrescens in the Macaronesian clade, with L. curtum and L. bituberculatum as
unrelated lineages. All likelihood and Bayesian analyses
of combined data confirmed the monophyly of the Euphorbia associated clade, with the semidegener–canum–
artemisiae clade as their closest relatives.
3.5. Populations associated with Euphorbia
Additional parsimony analyses of the euphorb associated species only, using L. Ôrumicis,Õ L. Ôpinicola,Õ and
L. curtum as outgroups, resulted in a basal L. lowei, with
L. bicaudatum as sister group to L. inarmatum, L.
ÔpilosumÕ and L. loweanum (Fig. 7). All clusters with
higher than 50% bootstrap support in this analysis also
occurred in the full parsimony analyses (Fig. 5). Three
of these strongly supported clusters represented deeply
divided populations of a polyphyletic L. Ôpilosum.Õ
B.H. Jordal et al. / Molecular Phylogenetics and Evolution 31 (2004) 554–571
563
Fig. 4. Maximum likelihood analyses of each gene partition, resulting in a single tree topology for each partition, identical to the Bayesian estimates.
Taxa with more than 30% missing data in one or both of the partitions were deleted from both partitions for improved parameter estimation and
comparison between the partitions. Parameter settings were as follow: CO1 (GTR + I + C), )ln ¼ 5554.79, rate matrix ¼ (1.32, 14.61, 1.05, 1.10, and
14.44) shape of gamma distribution 0.97, pinvar ¼ 0.60; EF-1a (TRN+I+C), )ln ¼ 3525.01, rate matrix ¼ (1.00, 3.30, 1.00, 1.00, and 11.62), shape of
gamma distribution 0.70, pinvar ¼ 0.55. Numbers on nodes show posterior probabilities P95%. Arrow points to the node defining a single origin of
Euphorbia feeding.
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B.H. Jordal et al. / Molecular Phylogenetics and Evolution 31 (2004) 554–571
Fig. 5. Strict consensus of 36 trees resulting from the unweighted parsimony analysis of all data; length 1922 steps, CI ¼ 0.37 (informative only),
RI ¼ 0.76. Arrows indicate collapsed nodes when taxa with >30 % missing data were excluded from this analysis. Stippled branches indicate the
placement of taxa which lacked EF-1a data. Bootstrap support values are shown above nodes, PBS values below (COI/EF/morphology). Host plant
genus is shown to the right of each specimen analysed (see Table 1 for further details).
Subsequent scrutiny of morphological characters revealed weak, but distinct differences in the surface
sculpture of the elytral declivity in these populations.
The ÔpilosumÕ C lineage differed from the A and B lin-
eages by lacking tubercles on declivital interstriae.
Lineage B may be distinguished from A by the lack of
tubercles on the first (median) declivital interstriae only.
Furthermore, all ÔpilosumÕ B specimens were collected
B.H. Jordal et al. / Molecular Phylogenetics and Evolution 31 (2004) 554–571
Table 3
Parsimony bootstrap support for three unstable clades as marked
in Fig. 6
Weighting scheme
clade Aa
clade Bb
clade Cc
1–1–1–1–1
1–1–1–0.5–1
1–1–1–0.2–1
1–1–1–0.1–1
78
76
77
66
na
56
67
66
na
66
82
80
1–0–1–1–1
1–0–1–0.5–1
1–0–1–0.2–1
1–0–1–0.1–1
72
76
77
74
na
<50
59
52
na
<50
73
80
1–1–1–1–0
1–1–1–0.5–0
1–1–1–0.2–0
1–1–1–0.1–0
77
81
75
74
na
na
<50
<50
na
na
<50
<50
1–0–1–1–0
1–0–1–0.5–0
1–0–1–0.2–0
1–0–1–0.1–0
69
77
72
71
na
na
<50
<50
na
na
<50
<50
Weighting scheme indicates the weight given to EF-1a exon – EF1a intron – COI 1st + 2nd positions – COI 3rd positions – morphology.
When clades did not occur in the most parsimonious trees, support was
not applicable (na).
a
Clade A: all Liparthrum except bartschti, ÔpinicolaÕ and ÔrumicisÕ;
includes L. nigrescens.
b
Clade B: all euphorb associated species plus artemisiae, canum,
and semidegener; excludes L. bituberculatum, L. colchicum, and L.
nigrescens.
c
Clade C: all Euphorbia associated species (monophyletic).
only from E. balsamifera, and may indicate ecological
divergence by an unreversed host switch. Only two
transitions to E. balsamifera were observed in the entire
clade of Euphorbia associates, in L. bicaudatum and
L. ÔpilosumÕ B, with no reversals. The two steps necessary to account for these transitions deviates significantly from a random distribution of host plant
associations based on 1000 random topologies (range
3–10, avg. 6.94, P ¼ 0:01).
565
4. Discussion
4.1. Phylogenetics of Macaronesian Liparthrum
Several major conflicts appeared from the analyses
under various optimality criteria and weighting schemes.
Resolution of the most basal lineages was particularly
difficult to achieve, as basal topologies were characterized by extremely shallow internodes with little or no
branch support. Given the high number of parsimony
informative characters available (total 437), the lack of
resolution in the earliest splits was not an artefact of
character limitation. The two partitions had also very
different substitution rates, which should provide sufficient differential support to various depths of the phylogeny. However, COI third positions showed evidence
of strong substitutional saturation and this may explain
some of the variable results. Likelihood and Bayesian
analyses are more likely (than parsimony) to compensate for high substitution rates and these analyses placed
L. nigrescens, L. bituberculatum, and L. curtum in more
basal positions compared to the unweighted parsimony
analysis. With an increased downweighting of COI in
the parsimony analyses, these results became largely
congruent with the combined Bayesian topology (mixed
model, all data, Fig. 6).
The most variable position in the tree involved the
placement of L. nigrescens, which differed across practically all analyses performed. Ambiguous placement of
this taxon was apparently not due to the observed biased
base frequencies in COI third positions, because the
variation between conspecific individuals was as large as
that between species. Not surprisingly then, Log determinant (LogDet) transformation of the data only confirmed our results from the Bayesian and weighted
parsimony analyses. To conclude, the uncertainties observed with respect to identifying the oldest Macaronesian lineage seems linked to a rather rapid divergence of
the first lineages on these islands.
Table 4
Bayesian estimates of nucleotide substitution parameters (GTR + C + I model), showing the mean and 95% credibility intervals
Parameters
CO1
ln
r-Gt
r-Ct
r-CG
r-AT
r-AG
r-AC
pA
pC
pG
pT
a
pinv
)5619.91
1.00
29.49
2.33
1.54
30.53
2.07
38.34
19.10
8.43
34.14
0.62
0.55
EF-1a
()5637.36, )5604.70)
(1.00, 1.00)
(10.53, 59.01)
(0.34, 5.54)
(0.44, 3.24)
(10.88, 47.20)
(0.60, 4.30
(35.37, 41.53)
(17.29, 20.97)
(6.91, 9.77)
(31.33, 36.46)
(0.42, 0.83)
(0.48, 0.60)
)3854.04
1.00
15.69
1.88
1.23
4.50
1.30
28.31
23.68
21.50
26.52
0.64
0.49
Combined
()3869.21, )3839.73)
(1.00, 1.00)
(9.44, 25.41)
(0.84, 3.57)
(0.65, 2.17)
(2.58, 7.87)
(0.66, 2.41)
(25.55, 31.41)
(21.04, 26.09)
(18.80, 23.85)
(23.69, 29.24)
(0.39, 0.95)
(0.33, 0.58)
Empirical base frequencies were used as a known prior at start of the analyses.
)9756.74
1.00
34.65
3.21
4.41
14.76
3.10
30.49
22.39
16.84
30.28
0.53
0.53
()9779.09, )9739.78)
(1.00, 1.00)
(17.54, 48.11)
(1.49, 5.33)
(2.18, 6.40)
(7.50, 22.20)
(1.37, 4.73)
(28.47, 32.84)
(20.67, 23.99)
(15.09, 18.86)
(28.63, 32.17)
(0.42, 0.70)
(0.47, 0.58)
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B.H. Jordal et al. / Molecular Phylogenetics and Evolution 31 (2004) 554–571
Fig. 6. The 50% majority rule consensus tree resulting from the Bayesian analysis of combined data for 52 taxa under a mixed model, consisting of
individual GTR + I + C models of DNA substitution for each of the COI and for EF-1a partitions, and a model for morphological transitions.
Numbers on nodes show posterior probabilities P 50%. Nodes marked by circled capitals (A, B, and C) refer to unstable internal branches tested
under various weighting schemes in the parsimony analyses. Host-plant families are indicated to the right of each taxon.
A more consistent result throughout the analyses,
and one which was independent of outgroup selection,
was the basal placement of L. ÔrumicisÕ and L. ÔpinicolaÕ
in the parsimony and Bayesian analyses. The placement
as the most basal clade (Figs. 5 and 6) even obtained
moderately strong support in these analyses and may
indicate the most basal lineage of Macaronesian Liparthrum. Given that a basal phylogenetic position is
important to the interpretation of character transformations, and that these basal species have previously
B.H. Jordal et al. / Molecular Phylogenetics and Evolution 31 (2004) 554–571
567
Fig. 7. Re-analysis of the combined data for species associated with Euphorbia only, using L. Ôrumicis,Õ L. Ôpinicola,Õ and L. curtum as outgroups.
Phylogram showing one of the eight most parsimonious trees of length 987, CI ¼ 0.52, RI ¼ 0.80. Bold bootstrap values highlight the two origins of
balsamifera-feeding in the Euphorbia associated clade. A map of the Canary Islands is depicted below the tree topology, with acronyms as described
in Fig. 3.
been infrequently sampled (they are undescribed species), their potential absence from the data matrix reminds us about some of the pitfalls of insufficient
sampling in phylogeny estimation (Emerson, 2002).
Many of the more derived relationships were strongly
supported by each of the data partitions and by the
combined data. The Madeiran L. semidegener is believed
to be closely related to the more widespread L. nigrescens (see Israelson, 1990); however, our data strongly
support a sister relationship to L. canum and L. artemisiae. Genitalia are also nearly identical in the three
species (cf. Figs. 1–11 in Israelson, 1990) further supporting our result. Liparthrum nigrescens grouped instead with L. colchicum and L. bituberculatum by the
separate COI and the combined unweighted parsimony
analyses, or in one of the most basal positions in the
likelihood or Bayesian analyses. The relationship of
these three laurel-feeding species to the legume- and
asterid-feeding species (L. semidegener, L. canum, and L.
artemisiae) was not strongly supported in the parsimony
analysis, and L. artemisiae and allies may be more closely related to the seven species associated with Euphorbia as suggested by all EF-1a analyses, the
combined weighted parsimony analysis, and in the
combined likelihood and Bayesian analyses. The relationship between the asterid- and euphorb-feeding spe-
cies is also supported by one shared genitalic character
(Appendix A: character 5, state 1).
Species breeding in Euphorbia were monophyletic in
all combined analyses and the separate analyses of EF1a. The lack of significant node support for this clade in
the combined parsimony analysis was apparently due
to incongruence with the COI partition, which placed
L. lowei nearer to the base of the tree. Although the
monophyly of this group in the parsimony analysis was
dependent on the addition of morphological characters,
monophyly was confirmed in likelihood and Bayesian
analyses of the combined molecular data. In fact, using
a mixed model which takes into account different evolutionary models for each of the three data partitions, a
significant posterior probability was obtained also for
this node. Our estimate thus confirms a phylogenetic
hypothesis which has been suggested in most taxonomic
treatments so far (Israelson, 1990; Schedl et al., 1959;
Wollaston, 1862). What was unexpected though, was the
paraphyly of L. loweanum with respect to L. ÔpilosumA.Õ The first species is very similar to L. lowei, as the
specific epithet implies, and rather dissimilar externally
to the Gran Canarian L. Ôpilosum-A.Õ Individuals of the
latter species are much larger than the L. loweanum
specimens, and have very long interstrial setae—a character shared with the other ÔpilosumÕ species. We note
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B.H. Jordal et al. / Molecular Phylogenetics and Evolution 31 (2004) 554–571
that the nodes connecting the two were shallow compared to the much longer terminal nodes. Hence we
cannot rule out long branch attraction in this case, and
we lean towards the much more conservative EF-1a
estimate of a monophyletic L. loweanum. However, the
deep coalescence between the two morphologically
identical Cape Verde populations (Santo Antao vs. Sao
Vicente) calls for a more detailed analysis of the phylogeography and possible cryptic geographical speciation in this species.
Divergence of the L. inarmatum–ÔpilosumÕ complex
into four highly supported clades was most evident in
the COI analyses, but only one (ML) or two (MP) individuals differed slightly in their positions based on the
EF-1a data. Such level of congruence is not commonly
observed within species, unless geographic or ecological
barriers restrict gene flow between populations (Avise,
2000). Population subdivision must also be maintained
during recent range expansion and this is seen in at least
one of the ÔpilosumÕ lineages (B), the distribution of
which overlaps with a second lineage in La Palma and
La Gomera (ÔpilosumÕ C). It seems likely that these
lineages represent multiple cryptic species, underscoring
the importance of sequencing multiple specimens per
species for detecting genetic differentiation (Emerson,
2002). Only individuals of L. inarmatum can easily be
distinguished morphologically from other populations
of the inarmatum–ÔpilosumÕ complex, by their shorter
interstrial setae. The ÔpilosumÕ lineages tested here have
very similar morphology, although the tiny differences
found may be distinct enough for describing all three
lineages as new species (Knizek, in prep.). Hence, this
study demonstrates the useful synergy between molecular and morphological data in modern taxonomy.
4.2. Cryptic host specialisation?
Previous reports on host records for Liparthrum
(summarised in Bright and Skidmore, 1997; Wood and
Bright, 1992) have given the impression that many
species use a wide range of host plants. However, recent
intensive collecting efforts, in particular from Euphorbia
plants, with subsequent analyses of genetic data, provide
a more rigorous test of diet breadth. Although the
sample size in this study is too low to be conclusive, the
significant correlation between specific Euphorbia associations and genetic clustering in the L. inarmatum–
ÔpilosumÕ complex is particularly striking. In addition, L.
lowei and L. bicaudatum seem to be more or less exclusively associated with E. lamarckii or E. balsamifera,
respectively (Fig. 7; Jordal, in prep). The combination of
host-plant correlation and deep coalescent in several
inarmatum–ÔpilosumÕ lineages creates a new scenario of
cryptic species distinguished partly by specific Euphorbia-host preference. Similar patterns are also seen in
other Macaronesian bark beetles, in particular Aphan-
arthrum, which also demonstrate a much higher degree
of specialisation to specific Euphorbia hosts than is apparent from literature records (Jordal and Hewitt, submitted). The diversification of Liparthrum and
Aphanarthrum due to host switching in the Euphorbia
alliance, rather than switching to other host-plant families available on these islands, suggest that beetles
adapted to life inside dead euphorbs are highly constrained in their host selection.
Less constrained host switching between plant genera
occurred in the canum–artemisiae clade, involving a
switch between genera in the Asteraceae. Although the
L. canum specimen used here was collected from Echium
(Asteridae: Boraginaceae), most records of this species
are from Argyranthemum and Asteriscus (Asteridae:
Asteraceae), and L. artemisiae is only found on Artemisia (Asteraceae). This relationship also demonstrates
another common pattern in herbivores, that the closest
relatives to extreme specialists are frequently less specialised species (e.g. Despres et al., 2002; Funk et al.,
1995; Kelley and Farrell, 1998; Kelley et al., 2000). This
pattern may also apply to the strictly pine feeding Liparthrum vs. L. Ôrumicis,Õ the latter of which has been
collected from several unrelated plant families.
The sister group relationships between species utilising the same or closely related host species represent a
case of host plant constraints for beetles breeding (and
feeding) in dead plant material. This is common in bark
beetle clades associated with living or recently dead host
plants (e.g., Beaver, 1979; Kelley and Farrell, 1998;
Noguera-Martinez and Atkinson, 1990), where the hostplantÕs defensive system is partially intact in partially
dead host plants or where defensive chemicals still remain. That beetles such as Liparthrum, living in older
and drier material, should demonstrate a similar degree
of specialisation is more surprising. However, high
abundance of suitable and widespread host plants foster
specialisation by increasing the probability of finding
new breeding resources (Beaver, 1979; Stevens, 1986).
The most specialised species in this study all breed in
woody plants which are among the most abundant in
their respective vegetational zones in Macaronesia (in
particular, Euphorbia, Artemisia, Laurus, and Pinus).
This could be one explanation for the apparent monophagy in many of the wood- and twig-boring species
of Liparthrum. On the other hand, the evident preference for specific Euphorbia species over others with
similar distributions suggests that plant chemistry possibly play an equally important role in shaping these
insect communities. Neither can host abundance explain
why some species apparently specialise on narrowly
distributed endemics, e.g., L. semidegener which breeds
only in dry twigs of Teline madeirense in Madeira (Israelson, 1990). Detailed comparative examination of
structural and chemical components in living and dead
plants can help resolve this issue.
B.H. Jordal et al. / Molecular Phylogenetics and Evolution 31 (2004) 554–571
4.3. Insular origins of species and populations
With the limited sample of continental species of Liparthrum we had available, there is little we can say about
monophyly of Macaronesian lineages or numbers of
colonisation events to the islands as a whole. However,
the Macaronesian species are not monophyletic given the
sister relationship between the Mediterranean L. colchicum and the Macaronesian L. bituberculatum (Figs. 3 and
5). The first species could have re-colonised the continent
from Macaronesia, but this is at odds with the phylogeographical history of Laurus, their sole host plant. The
origin of the Macaronesian Laurus azorica was quite recent judged by its nested position within the continental
L. nobilis (Arroyo-Garcıa et al., 2001; Emerson, 2003); if
no ancient host switching has occurred within the laurel
associated clade of beetles, L. bituberculatum has also
most likely colonised the islands recently. A recent expansion in this species is further suggested by almost no
genetic differentiation among three islands.
High colonisation potential seems to be a general
trait in Liparthrum since most species are found on three
or more islands (Bright and Skidmore, 1997; Wood and
Bright, 1992). Some species are even found on entire
archipelagos and thus are much less restricted in their
distribution than many other Macaronesian insects
(Juan et al., 2000). Dispersal capability is particularly
well demonstrated by the multiple recent expansions
observed in L. inarmatum and two L. ÔpilosumÕ lineages
(B and C, see Figs. 4 and 6), and at least L. curtum
provides further evidence for the direction of dispersal
being from the western Canary Islands to Madeira. This
pattern is not common in the insect groups studied so
far, but is quite frequent in plants (Bohle et al., 1996;
Panero et al., 1999). Many previous studies (Juan et al.,
2000) have focused on flightless insects, however, which
may explain this rarity in insects, and recent studies
(Jordal and Hewitt, submitted; Percy, 2003) have found
a pattern similar to that in Liparthrum.
While many Macaronesian Liparthrum have expanded their ranges in recent times, it is not very likely
that most species are recent colonisers from the continent. The great sequence divergence observed within
many species indicates prolonged habitation of Macaronesia for each species (see Table 2, cf. Fig. 4). Liparthrum nigrescens, L. curtum, and L. artemisiae fit this
description well, but the largest intra-specific divergence
was found in L. loweanum. This species is found on most
of the Cape Verde islands (Wood and Bright, 1992) and
the deep split between the Santo Antao and Sao Vicente
populations (15.5% ML corrected COI divergence)
suggest presence on these islands for 6.7 Mya (assuming
approximately 2.3% mtDNA divergence rate in arthropods: Brower, 1994). It is interesting to observe that
L. loweanum may be related to an ancestral lineage of
L. ÔpilosumÕ from Gran Canaria, suggesting common
569
routes of dispersal for insect and host plant. The Cape
Verdian host plant, E. tuckeyana, is closely related to the
eastern Canary islands E. regis-jubae (based on reanalysis of Molero et al.Õs (2002) ITS data), and similar
routes of dispersal has also been suggested for Echium
plants (Bohle et al., 1996).
Based on maximum ML-corrected COI divergences,
Macaronesian Liparthrum had a most recent common
ancestor as old as the islands (20 Mya). Even the
youngest part of the Macaronesian radiation—the Euphorbia associated clade—is at least 11.3 Mya using the
2.3% arthropod substitution rate. Taken together with
considerable interspecific variation at the EF-1a locus,
our genetic data argue against a recent origin of Liparthrum, as well as against a recent origin of the island
radiation(s). Furthermore, the high ratio of specific to
interspecific branch lengths in the oldest lineages supports the view of a relict distribution of ancient lineages
(Wood, 1986). Extinctions may be a plausible explanation for the distant relationship observed among the
majority of lineages, but an alternative explanation is
that many species still remain uncollected, perhaps due
to their small size and that they breed in old woody
tissues of little economic importance (Israelson, 1990).
Intensive collecting efforts during the last 30 years have
resulted in at least 11 new species (Israelson, 1990;
Knizek, in prep), and the addition of seven of these (L.
semidegener, L. canum, L. Ôpinicola,Õ L. Ôrumicis,Õ L.
ÔpilosumÕ A, B, and C) reduced internal branch lengths
in several clades (cf. Figs. 4 and 6). More undescribed
species may await discovery and could help to better
resolve the basal nodes in the phylogeny. However, this
will not change our conclusion that the lack of morphological distinctiveness in Liparthrum is not due to a
recent origin and radiation of the group.
Acknowledgments
We thank Milos Knizek for assistance on the taxonomy of Liparthrum, Maria Lombardero, M. Arechevaleta, Massimo Faccoli for providing samples,
Roberto Jardim and Pedro Oromi for help with permits
and logistics, and the Cabildos of Fuerteventura, Lanzarote, Gran Canaria, Tenerife, La Gomera, La Palma,
and El Hierro for collecting permits. This research was
funded by a Norwegian Research Council SUP grant
NFR-128388/420 to Kirkendall and a Marie Curie
Fellowship HPMF-CT2001-01323 to Jordal.
Appendix A. Morphological characters (informative only)
used in the combined phylogenetic analyses of all data
1. Strial punctures: not impressed (0); strongly impressed (1)
570
B.H. Jordal et al. / Molecular Phylogenetics and Evolution 31 (2004) 554–571
2. Interstrial setae: scale-like (0); hair-like (1); scattered
bristles (2); absent (3)
3. Posterior end of elytra: parallel to narrowly rounded
(0); dilated (1)
4. Apodemes of aedeagus: long and slender (0); very
short (1)
5. Tegmen of aedeagus: simple ring (0); very thin, with
narrowly pointed manubrium (1); not sclerotized dorsally (2); thick, manubrium wide at base (3)
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