Molecular Phylogenetics and Evolution 64 (2012) 331341

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Molecular Phylogenetics and Evolution

journal homepage: www.elsevier.com/locate/ympev

Phylogenetics and evolution of host-plant use in leaf-mining sawies

(Hymenoptera: Tenthredinidae: Heterarthrinae)
Sanna A. Leppnen a,, Ewald Altenhofer b, Andrew D. Liston c, Tommi Nyman a,d
a

Department of Biology, University of Eastern Finland, P.O. Box 111, FI-80101 Joensuu, Finland
Etzen 39, A-3920 Gro Gerungs, Austria
c
Senckenberg Deutsches Entomologisches Institut, Eberswalder Strae 90, 15374 Mncheberg, Germany
d
Institute of Systematic Botany, University of Zrich, Zollikerstrasse 107, CH-8008 Zrich, Switzerland
b

a r t i c l e

i n f o

Article history:
Received 27 October 2011
Revised 21 March 2012
Accepted 6 April 2012
Available online 17 April 2012
Keywords:
Coevolution
Host shifts
Insectplant interactions
Heterarthrinae
Leaf-mining insects
Speciation

a b s t r a c t
The habit of mining within leaves has evolved convergently in numerous plant-feeding insect taxa. Many
leaf-mining groups contain a large number of species with distinct feeding preferences, which makes
them highly suitable for studies on the evolutionary history of host-plant use and on the role of niche
shifts in speciation. We aimed to clarify the origin, classication, and ecological evolution of the tenthredinid sawy subfamily Heterarthrinae, which contains c. 150 leaf-mining species that collectively feed on
over 20 plant genera around the world. For this, we reconstructed the phylogeny of representative
heterarthrine species and diverse outgroups from the superfamily Tenthredinoidea on the basis of
DNA sequence data collected from two mitochondrial (CoI and Cytb) and two nuclear (EF-1a and NaK)
genes. Thereafter, we inferred the history of niche diversication within Heterarthrinae by plotting larval
host-plant associations on the trees, and by contrasting a time-calibrated leaf-miner phylogeny with the
phylogeny of their host plants. The results show that: (1) heterarthrine leaf-miners constitute a monophyletic group that arose from external-feeding blennocampine lineages within the Tenthredinidae c.
11080 million years ago; (2) heterarthrines generally radiated well after their host taxa, and extant
host-plant associations therefore result from a combination of host conservatism and occasional shifts
among available plant taxa; and (3) diversication in Heterarthrinae apparently occurs by multiple
mechanisms, including sympatric or allopatric ecological speciation, non-ecological allopatric speciation,
and possibly allochronic speciation. Overall, both present and historical host-use patterns within the Heterarthrinae exhibit striking similarities to patterns found in co-occurring herbivore taxa.
2012 Elsevier Inc. All rights reserved.

1. Introduction
The habit of mining within leaves has evolved several times
convergently in different lineages of Diptera, Lepidoptera, Coleoptera, and Hymenoptera, the most diverse groups being found within Lepidoptera and in the dipteran family Agromyzidae (Connor
and Taverner, 1997; Hespenheide, 1991; Sinclair and Hughes,
2010). The reasons for the multiple origins of leaf-mining may be
that mines shelter the larvae from environmental stress and natural enemies, and that feeding inside leaves enables larvae to avoid
plant defenses (Connor and Taverner, 1997). Many leaf-mining insect taxa constitute excellent model groups for phylogeny-based
studies on the evolutionary history of insectplant associations:
Leaf-mining groups often contain numerous species that feed on
Corresponding author. Fax: +358 13 251 3590.
E-mail addresses: sanna.a.leppanen@uef. (S.A. Leppnen), ewald.altenhofer@
aon.at (E. Altenhofer), andrew.liston@senckenberg.de (A.D. Liston), tommi.nyman
@uef. (T. Nyman).
1055-7903/$ - see front matter 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.ympev.2012.04.005

different hosts, while, at the same time, individual species tend

to specialize on single plant species or on restricted groups of closely related plants (Hespenheide, 1991; Menken et al., 2010;
Smith, 1979). This makes it possible to study, for example, patterns
of coevolutionary relationships and the relative timing of diversication in interacting miner and plant groups, and to evaluate the
role of host-plant shifts in insect speciation (Lopez-Vaamonde
et al., 2003, 2006; Scheffer et al., 2007; Winkler et al., 2009).
We studied the origin, phylogeny, and ecological evolution of
the less-studied hymenopteran leaf-miners in the subfamily Heterarthrinae. Heterarthrinae belongs to the Tenthredinidae, which is
the largest family in the ancestrally herbivorous symphytan (=sawy) lineages of Hymenoptera (Davis et al., 2010; Viitasaari, 2002).
As currently dened, Heterarthrinae includes 38 genera and
approximately 230 species (Goulet, 1992; Taeger et al., 2010).
However, the origin and exact composition of Heterarthrinae is
ambiguous, as is reected by clearly contrasting traditional classications: Most recent authors have considered leaf-mining species
(in the genus Heterarthrus and in the tribe Fenusini, which includes

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S.A. Leppnen et al. / Molecular Phylogenetics and Evolution 64 (2012) 331341

the rest of the genera) to constitute a monophyletic group within

Heterarthrinae, but they have also included external-feeding
groups (particularly leaf-skeletonizers of the genera Caliroa and
Endelomyia) as the tribe Caliroini in the same subfamily (Goulet,
1992; Smith, 1971; Taeger et al., 2010; Viitasaari, 2002). By contrast, others have proposed that the leaf-mining lineages of Heterarthrinae are polyphyletic, so that leaf-mining would have
originated independently in the genus Heterarthrus and in the tribe
Fenusini, the latter of which would be included in the ecologically
diverse tenthredinid subfamily Blennocampinae (Pschorn-Walcher
and Altenhofer, 1989). However, our phylogenetic results (see below) indicate that Heterarthrus and the fenusines constitute a
monophyletic group separated from the polyphyletic caliroine genera, and so we will hereafter use Heterarthrinae in a sense that includes only the leaf-mining taxa. In this narrow sense,
Heterarthrinae encompasses 152 described species in 29 genera
(Supplementary Table 1).
Heterarthrine leaf-miners are highly suitable for research on
food-web structure and assembly because of their taxonomic and
ecological diversity, wide geographic distribution, and the abundance of many species (Supplementary Table 1). Heterarthrus is
Palearctic with two invasive species occurring in North America
(Digweed et al., 2009; Humble, 2010; Smith, 1971), but species
belonging to the fenusine genera can be found in Europe, Asia,
and North and South America (Smith, 1976). Most heterarthrines
are relatively specialized in their host-plant use and typically feed
on one to a few congeneric or confamilial plant species (PschornWalcher and Altenhofer, 1989; Smith, 1979; Taeger et al., 1998),
but their collective host range encompasses over 20 plant genera
around the world (Altenhofer, 2003; Smith, 1971). Most of the
hosts are woody species in the families Rosaceae, Ulmaceae, Betulaceae, Fagaceae, Nothofagaceae, Salicaceae, Malvaceae, Sapindaceae, and Oleaceae, but numerous heterarthrines also feed on
herbaceous plants in Rosaceae and Geraniaceae.
In the present work, our main aims were to: (1) elucidate the
origin, phylogenetic position, and age of heterarthrine sawies
within the family Tenthredinidae; (2) clarify the internal phylogeny and classication of Heterarthrinae; and (3) study the
evolutionary history of host-plant use in heterarthrine leaf-miners.
In order to answer the rst two questions, we used Bayesian,
maximum-likelihood, and maximum-parsimony methods to
reconstruct the phylogeny of representative heterarthrine and outgroup taxa based on sequence data collected from two mitochondrial and two nuclear genes. The third question was tackled by
plotting current host-plant associations on the resulting phylogenetic trees, and by contrasting a time-calibrated sawy phylogeny
with a phylogeny of representative host and non-host plant taxa.
The results provide new information on the origins of leaf-mining
within the Hymenoptera, and shed light on factors that lead to speciation and to diversication of host-plant use in leaf-mining
insects.

2. Methods
2.1. Taxon sampling
Our phylogenetic analyses concentrated on heterarthrine species for which data on host-plant use are available (Supplementary
Table 1). The 39 ingroup species included in our study represent a
large proportion of the leaf-mining genera and nearly all of the
European species (Supplementary Tables 1 and 2). Two to four
specimens from different locations were used for widely distributed miner species that use more than one plant species as a host,
so the total number of ingroup exemplars is 58. In order to clarify
the age and phylogenetic position of the heterarthrine leaf-miners,

we extensively sampled taxa representing all tenthredinid

subfamilies, as well as representatives of all non-tenthredinid families of the superfamily Tenthredinoidea (i.e., Blasticotomidae,
Argidae, Pergidae, Cimbicidae, and Diprionidae). Hence, the total
number of specimens included in the phylogenetic analyses is 88
(Supplementary Table 2).
2.2. DNA extraction, PCR amplication, and sequencing
We extracted DNA from larval and adult samples stored in
99.5% ethanol at 20 C by using the DNeasy Tissue Kit (Qiagen,
Valencia, CA). Sequence data were collected from the mitochondrial genes Cytochrome oxidase I (CoI) and Cytochrome b (Cytb),
and the nuclear genes Elongation factor-1a (EF-1a; two exons of
the F2 copy) and Sodiumpotassium adenosine triphosphatase
(NaK). PCR amplication and sequencing of CoI, Cytb, and EF-1a
were done as described earlier (Nyman et al., 2000, 2006a). To amplify a portion of the NaK gene, we initially used the primers NaKfor2 and NaKrev2 (Danforth et al., 2004) for a subset of the taxa,
and then developed the sawy-specic primers NaKfor10 and NaKrev1000 (Table 1) for subsequent amplications. Each 50-ll PCR
reaction consisted of 1 Qiagen reaction buffer with MgCl2 added
to a nal concentration of 2.5 mM, 0.2 lM of each primer, 0.15 mM
of each dNTP, and 1.5 units of Taq DNA polymerase. The NaK PCR
program consisted of an initial denaturing step at 94 C for
5 min, followed by 40 cycles of 1 min denaturing at 94 C, 45 s
annealing at 55 C, and 2 min extension at 72 C; the last cycle
was followed by a nal 5 min extension step. Double-stranded
PCR products were puried using the QIAquick PCR Purication
Kit (Qiagen), and sequenced in both directions with the BigDye
Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster
City, CA) and an ABI 3730 automated sequencer (Applied Biosystems). The internal primers NaKfor450, NaKrev455, NaKfor470,
and NaKrev475 were used to conrm the sequences as needed
(Table 1).
CoI (810 bp), EF-1a (501 + 276 = 777 bp), and NaK (997 bp) sequences did not vary in length, and they were read, edited, and
aligned using Sequencher 4.8 (Gene Codes Corp., Ann Arbor, MI).
Due to two different three-base insertions in a few species, Cytb sequence lengths varied between 718 and 721 bp; these sequences
were therefore aligned by using ClustalW (Larkin et al., 2007) in
Mesquite version 2.71 (Maddison and Maddison, 2009), and the nal 724-bp alignment was checked manually. In total, the fourgene alignment contains 3308 base pairs (Table 2). Most species
have sequences from all four genes, but 24 specimens are missing
one gene, nine specimens are missing two genes, and four specimens have sequences from only a single gene (Supplementary Table 2). All sequences have been deposited in GenBank under
accession numbers CoI: JX090726 - JX090784 Cytb: JX090785 JX090855 Ef1a: JX090856 - JX090924 NaK: JX090925 - JX091002.
Table 1
Primers used to amplify and sequence the nuclear NaK gene in the studied sawy
species. PCR primers are marked with an asterisk. NaKfor2, NaKrev2 (Danforth et al.,
2004), NaKfor10, and NaKrev1000 were used for amplication and sequencing, other
primers were used as internal sequencing primers as needed (for and rev denote
forward and reverse primers, respectively). Numbers of the newly designed primers
indicate approximate binding sites in relation to NaKfor2.
Primer

Sequence (50 30 )

NaKfor2
NaKfor10
NaKfor450
NaKrev455
NaKfor470
NaKrev475
NaKrev2
NaKrev1000

GCS TTC TTC TCB ACS AAC GCC GTY GAR GG

AAC GCC GTY GAR GGT ACY GCR AAG GG
ATG ACC GTY GCY CAY ATG TGG TT
AAC CAC ATR TGR GCR ACG GTC AT
ATG TGG TTY GAY AAY CAR ATY ATC GA
TCG ATR ATY TGR TTR TCR AAC CAC AT
ACC TTG ATR CCG GCY GAW CGG CAC TTG GC
CTG GGW GGA TCR ATC ATG GAC AT

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Table 2
Basic information on the dataset and separate genes used in Heterarthrinae
phylogeny reconstruction: Number of characters (Ch), variable sites (V), parsimony
informative sites (PI), number of individuals and species (in parentheses) covered in
the full data (TC-f) and within the ingroup (TC-i).
Gene

Ch

PI

TC-f

TC-i

Full data
CoI
Cytb
Ef-1a
NaK

3308
810
724
777
997

1599
437
479
277
406

1431
382
422
242
385

79
70
69
77

49
48
43
52

(64)
(56)
(59)
(61)

(34)
(35)
(33)
(36)

2.3. Phylogeny reconstruction

2.3.1. Leaf-miners
We performed Bayesian phylogenetic analyses in MrBayes v.
3.1.2 (Ronquist and Huelsenbeck, 2003) and in BEAST v. 1.5.2
(Drummond and Rambaut, 2007). Prior to the analyses, we used
jModelTest v. 0.1.1 (Posada, 2008) to select the least complex substitution model for each gene. Likelihood calculations were based
on trees calculated by PhyML (Guindon and Gascuel, 2003) within
jModelTest, and model selection was done on the basis of the
Akaike Information Criterion (AIC). A GTR + I + C model of substitution was found to be optimal for all genes except NaK; however,
since the model selected for NaK (TIM2 + I + C) is not implemented
in MrBayes, we also used for this gene the next more parameterrich model (GTR + I + C). In MrBayes, we used default priors, and
each of the four partitions was allowed to have its own unlinked
substitution model. We ran two parallel runs having six incrementally heated chains (t = 0.15) for 10 million generations, while sampling trees from the current cold chain every 100 generations. We
discarded 40,001 trees sampled prior to reaching chain stationarity
as a burn-in from both runs, and the remaining 120,000 trees were
used to calculate a consensus tree showing all compatible groupings. MrBayes phylogenies were rooted with the blasticotomid
Blasticotoma liceti (Klug), following Schulmeister (2003) and Davis
et al. (2010).
BEAST was used to estimate the phylogeny and diversication
times of leaf-mining sawies using a Bayesian relaxed molecularclock method (see Drummond et al., 2006). The topologically
unconstrained analysis allowed a separate GTR + I + C4 substitution model for each gene, and employed the uncorrelated lognormal prior model of rate change, a Yule prior process to model
speciation, and automatic tuning of operators. We calibrated the
tree by placing a uniform 135155 million-year prior on the age
of the split between Tenthredinidae and the Diprionidae + Cimbicidae clade, based on the oldest known tenthredinid Palaeathalia
laiyangensis Zhang from the late Jurassic or early Cretaceous (Darling and Sharkey, 1990; Labandeira, 1994; see also Grimaldi and
Engel, 2005). Two independent analyses were run for 60 million
generations, while trees and parameter estimates were sampled
once every 1000 generations. Log les of the runs were inspected
in Tracer v. 1.4.1 (Rambaut and Drummond, 2007), to conrm
reaching chain stationarity and adequate effective sample sizes
(ESSs) for the estimated parameters. The tree les were then combined in LogCombiner v. 1.5.2 (part of the BEAST package), with the
burn-in set to 6001 trees (i.e., 6,000,001 generations) and resampling trees every 3000 generations. The remaining 36,000 trees
were thereafter used to generate a maximum clade credibility
(MCC) tree with mean node heights using TreeAnnotator v. 1.5.2
(part of the BEAST package).
Phylogenies were also reconstructed using maximumlikelihood (ML) and maximum-parsimony (MP) approaches. ML
and MP trees were rooted using Blasticotoma liceti as in the
MrBayes analyses described above. ML analyses were performed
using RAxML version 7.2.8 (Stamatakis et al., 2008) at the CIPRES

333

Web portal (Miller et al., 2010) using two different partition

schemes. In the rst analysis, a separate GTR + I + C model was
employed for each gene. In the second ML analysis, the dataset
was rst partitioned into genes and then into codon positions
1 + 2 and 3 within each gene (i.e., eight partitions in all). In both
cases, node supports were evaluated based on 100 bootstrap replicates. MP searches based on Nixons (1999) parsimony ratchet
method were performed by using PAUPRat (Sikes and Lewis,
2001) in conjunction with PAUP version 4.0b10 (Swofford,
2003). The ratchet searches employed the incremental reweighing
proportion scheme of Nyman et al. (2006a). Tree searches were
rst done with unweighed data, and then weighing transversions
3:1 over transitions; in both cases, gaps were treated as missing
data. Clade support was evaluated in PAUP by bootstrapping
(Felsenstein, 1985) the datasets 100 times while excluding uninformative characters. The heuristic tree searches implemented tree
bisection and reconnection (TBR) branch swapping, twenty random addition sequences per pseudoreplicate, and no maxtrees
limit.
As an additional exploration of the sawy sequence dataset, we
used the PhyDesign online application developed by LpezGirldez and Townsend (2011) to generate net phylogenetic informativeness (PI; Townsend, 2007) proles for each gene through
time. PI curves were also estimated separately for codon positions
1 + 2 and 3 within each gene, as well as for each codon position
within each gene. PI plots in PhyDesign were based on the aforementioned MCC chronogram calculated in BEAST, and implemented the HyPhy program (Pond et al., 2005) to calculate site
rates based on a HKY85 model of substitution (i.e., nucleotide frequencies estimated separately for each partition, transition/transversion rate ratio set to j = 2).
2.3.2. Plants
We used BEAST to construct a time-calibrated genus-level phylogeny for selected plants on the basis of matK, 26S, 18S, rbcL, and
atpB sequences. Sequences for genera utilized by heterarthrine
leaf-miners were mostly obtained from Burleigh et al. (2009), but
for Potentilla, Rosa, Rubus, Geum, Ulmus, Nothofagus, and Phillyrea
we used GenBank sequences (Supplementary Table 3). We also
included Spiraea (Rosaceae) and Olea (Oleaceae) in the analyses,
because these had extensive sequences in the Burleigh et al.
(2009) dataset, while most of their confamilial GenBank representatives were missing at least two of the focal genes (in essence,
Spiraea and Olea were used to facilitate the correct grouping of their
respective families). In addition, we randomly selected one representative from each angiosperm order, as well as all outgroup species, from the analysis of Wang et al. (2009), with the aim of
conrming the correct place of our focal plant genera in the tree.
We used the original Burleigh et al. (2009) sequence alignments
for 26S, rbcL, and atpB, but made minor corrections to the matK
and 18S alignments (Supplementary Table 3). We calibrated the
phylogeny by setting a uniform 102111 million-year prior on
the age of the split between Rosales and the Cucurbitales + Fagales
clade, following the BRC-1 age estimate of Wang et al., 2009; (see
Table 1 in that article). We also constrained Platanus and Trochodendron to the root, as they were in Wang et al. (2009), to achieve
basic comparability with the tree in their paper. The starting tree
was randomly generated and manually edited in TreeEdit version
1.0a10 (Rambaut and Charleston, 2002) to conform with the aforementioned constraints placed on time and topology. In the analysis,
a separate GTR + I + C4 model of substitution was allowed for each
gene, and an uncorrelated lognormal prior model of rate change, a
Yule prior process to model speciation, and automatic tuning of
operators were applied. Two separate analyses were run for
20 million generations, while trees and parameter estimates were
sampled once every 1000 generations. Following log le inspection

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S.A. Leppnen et al. / Molecular Phylogenetics and Evolution 64 (2012) 331341

in Tracer, the tree les were fused in LogCombiner. The rst 2001
trees from each le were removed as a burn-in, and an MCC tree
showing mean node heights was produced in TreeAnnotator on
the basis of the remaining 36,000 post-stationarity trees.
2.4. Evolution of host-plant use and larval habits
Host plants of the studied heterarthrine species are according to
Altenhofer (2003), Goulet (1992), and Taeger et al. (1998) (the hosts
of the undescribed North American Scolioneura species are unknown). In all, the leaf-miners in our study feed on over 36 plant
species from 15 different genera belonging to ten families (Supplementary Table 2). Host-plant genera for each heterarthrine species
were coded as unordered multistate characters, and ancestral associations were reconstructed by Mk1-model maximum-likelihood
optimization across the ingroup parts of the BEAST MCC tree in Mesquite v. 2.71 (Maddison and Maddison, 2009). Larval feeding habits
(external feeder/leaf-miner/gall-inducer/shoot-borer/leaf-roller)
were coded for all included species and then similarly reconstructed
across the whole MCC tree. In both cases, the trees were pruned so
that each ingroup species was represented by a single individual. In
order to infer how phylogenetic uncertainty inuences the reconstructions, both analyses were repeated across 1000 trees sampled
from the posterior distribution during the BEAST run. These were
obtained by using LogCombiner to select every 36th tree from the
original 36,000-tree sample, and the trees in the thinned sample
were then pruned in Mesquite for each analysis as described above.
Reconstructions were performed using the Trace characters over
trees option in Mesquite and summarized on the pruned MCC trees;
pie diagrams at nodes show counts of trees with uniquely best
states, with the decision threshold set to T = 2.
Phylogenetic conservatism in the use of host genera across the
pruned MCC tree was inferred by the parsimony-based permutation tail probability (PTP) test (Maddison and Slatkin, 1991) in
Mesquite, using 1000 permuted replicates of the host character.
Conservatism in host use was also tested in a likelihood framework
by estimating Pagels (1999) lambda (k) parameter using the tDiscrete function in the package geiger (Harmon et al., 2008) in R v.
2.14.0 (R Development Core Team, 2009). In this case, statistical
signicance was evaluated by a likelihood ratio test, in which the
likelihood of the model with the estimated value of lambda was
contrasted with the likelihood of a model assuming no phylogenetic signal (i.e., lambda set to k = 0).
Temporal correspondence between the radiations of heterarthrine leaf-miners and plants was evaluated by rst identifying those
nodes on the ingroup part of the MCC tree that have posterior
probabilities over 50% and encompass species occurring on multiple different plant genera (thus requiring a shift either at the node,
or after it in time), and then plotting the ages of those nodes
against the crown-group ages of the plant clades that contain all
host genera utilized by the species in the sawy clade. The existence of statistically signicant topological (branching-order) congruence between the leaf-miner and plant phylogenies was tested
by rst estimating the maximum number of cospeciation events
needed for reconciling the miner and plant phylogenies in TreeMap
3b (Charleston, 2011), and then comparing this estimate to the distribution of corresponding values obtained by randomizing the
miner tree 100 times while keeping minerplant associations unchanged (Charleston and Robertson, 2002). In order to reduce the
computational complexity of the test, the plant phylogeny was
pruned to include only plant genera that are used by leaf-miners,
and the leaf-miner tree was simplied by pruning monophyletic
clades of species sharing a given host genus down to a single species. Analyses were performed using 25 starts from random maps
and heuristic searches for up to 25 generations.

3. Results
3.1. Sawy and plant phylogenies
Phylogenetic trees produced by Bayesian inference in MrBayes
(Fig. 1) and BEAST (Fig. 2A), and maximum-likelihood analyses in
RAxML (Supplementary Fig. 1), are relatively congruent and wellsupported. The results indicate that the heterarthrine leaf-miners
originated from within the diverse and most likely para- or polyphyletic subfamily Blennocampinae. According to the BEAST MCC
tree, heterarthrines constitute a monophyletic clade that arose c.
100 million years ago. A monophyletic Heterarthrinae is also
recovered by the eight-partition ML analysis (Supplementary
Fig. 1B) but, as in the MCC tree, the support for the inclusion of
the South American Notofenusa species and the Mediterranean endemic Silliana lhommei in Heterarthrinae is low. In the MrBayes
tree (Fig. 1) as well as in the gene-partitioned ML tree (Supplementary Fig. 1A), S. lhommei is (weakly) grouped with the gall-inducing
outgroup species Hoplocampoides xylostei. All trees indicate that
the caliroine leaf-skeletonizers Endelomyia and Caliroa form a
polyphyletic group inside Blennocampinae, instead of belonging
to the Heterarthrinae, although these two genera are weakly
grouped together in the gene-partitioned ML tree (Supplementary
Fig. 1A).
Maximum-parsimony analyses of the sequence data produced
more complex results. Ratchet runs with unweighed data produced
18 equally parsimonious trees of 13,625 steps, while the analysis in
which transversions were weighed three times more than transversions resulted in ten shortest trees of 26,802 steps. The strict
consensus trees of these analyses (Supplementary Fig. 2) present
some marked differences as compared to the model-based trees:
In the unweighed consensus tree, Endelomyia aethiops, Hoplocampoides xylostei, and Phymatocera aterrima are intermixed with
leaf-miners (Supplementary Fig. 2A). Weighing transversions three
times more than transitions resulted in a mostly monophyletic
Heterarthrinae, with the exclusion of Silliana and Notofenusa (Supplementary Fig. 2B). However, bootstrap support values for these
groupings in the MP trees are generally low.
Interrelationships among genera and species within Heterarthrinae are generally consistent and strongly supported in all
Bayesian and ML analyses; the main disagreements concern
weakly supported groupings such as the placement of the Profenusa thomsoni + pygmaea + lucifex clade, the exact locations of
Fenusella nana and Profenusa japonica, and the branching order
within the genus Parna (Figs. 1 and 2A). Many genus-level groups
within Heterarthrinae found in the Bayesian and ML analyses are
present and strongly supported also in the strict consensus trees
from the MP runs (Supplementary Fig. 2).
The trees mostly agree with the traditional generic assignments
of heterarthrine species, but also reveal some apparent misclassications: As currently dened, Fenusa is clearly paraphyletic with
respect to Fenella, and Profenusa is polyphyletic because P. japonica
is distantly related to the other species in the genus (Profenusa
comes out as monophyletic only in the unweighed MP analysis,
and with low bootstrap support). All analyses also strongly reject
the traditional Fenusini/Heterarthrini (=Heterarthrus) division of
leaf-miners, because Heterarthrus is strongly grouped as the sister
clade of Metallus, i.e., well inside the tribe Fenusini. Conspecic
individuals collected from different locations and/or hosts form
strongly supported monophyletic clades, with the exception of
Scolioneura betuleti and S. vicina individuals, which are intermixed
in the tree.
The phylogenetic informativeness proles indicate that all
four genes are informative across the whole MCC tree, and that
peak informativeness is reached within the time frame of the

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335

Fig. 1. Phylogeny of leaf-mining sawies from the subfamily Heterarthrinae and selected outgroups from Tenthredinidae and the ve other families of Tenthredinoidea based
on a Bayesian phylogenetic analysis in MrBayes. Tenthredinid subfamilies are denoted to the right of the tree. Numbers above branches are Bayesian posterior probabilities
(%), while numbers below branches show bootstrap proportions (%) from a maximum-likelihood analysis in RAxML (see Supplementary Fig. 1A). Hyphens in parentheses
below branches denote clades that are not present in the maximum-likelihood tree.

Heterarthrinae radiation (Fig. 3). Highest net PI is obtained for

Cytb, followed by CoI and NaK, while the apparently slow-evolving
EF-1a gene contains comparatively little information. As expected,
the largely degenerate third positions within codons contribute the
majority of net PI in all four genes, and net informativeness peaks
earlier (when going back in time) in the mitochondrial genes than
it does in the nuclear ones.
The topology of our genus-level plant phylogeny, as well as inferred divergence times among clades (Fig. 2B and Supplementary
Fig. 3), are approximately the same as in the original sources of the

sequence data (cf. Burleigh et al., 2009; Wang et al., 2009). Minor
differences to some prior analyses do, however, occur (e.g., Potter
et al., 2007 obtained a different relative placement of Geum and
Rubus, but with weak support), but such discrepancies do not
inuence our general inferences below. Deep divergences in our
tree tend to be slightly older than those found in some analyses
(cf. Bell et al., 2010; Fiz-Palacios et al., 2011; Magalln, 2010;
Wikstrm et al., 2001), but 95% highest posterior density intervals
of node ages generally overlap with those of corresponding splits
in previous trees.

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S.A. Leppnen et al. / Molecular Phylogenetics and Evolution 64 (2012) 331341

Fig. 2. Relaxed molecular-clock phylogeny of heterarthrine leaf-mining sawies and representative outgroup taxa of Tenthredinidae and ve other tenthredinoid families (A),
with simplied phylogeny of heterarthrine host-plant genera (B). Numbers above branches are posterior probabilities (%), numbers on nodes refer to split ages used in Fig. 4,
and gray bars show 95% highest posterior density intervals for node ages. Branch colors in the ingroup parts of (A) show ML reconstructions of ancestral host-plant genera
(see B); nodes and subtending branches were assigned a generic color only when the relative probability of the most likely plant genus at a given node exceeded 90% (see
Supplementary Fig. 6A). Larval feeding habits of exemplar species in (A) are indicated by different symbols in front of the species names (see legend). The full plant phylogeny
is included as Supplementary Fig. 4.

3.2. Evolution of larval feeding modes and host-plant use

ML reconstruction of larval feeding modes on the BEAST MCC
tree (Supplementary Fig. 4A) indicates a single adoption of the
leaf-mining habit coinciding with the origin of Heterarthrinae:
the relative likelihood of leaf-mining at the ingroup node is

99.6%, while the immediately subtending node receives an over

95.6% probability of being an external feeder. This inference is,
however, less clear when tracing larval habits across 1000 postburnin trees, in which case the uncertainty about the placement
of Silliana lhommei inuences the node below the ingroup (Supplementary Fig. 4B). In both reconstructions, a separate origin of

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S.A. Leppnen et al. / Molecular Phylogenetics and Evolution 64 (2012) 331341

1562

160

140

120

100
80
60
Millions of years ago

40

20

1093.4
937.2
781
624.8
468.6
312.4
156.2

180

1269
1057.5
846
634.5
423

140

120

100

80

60

40

20

CoI pos12

NaK pos12

EF1a pos12

Cytb pos3

CoI pos3

NaK pos3

EF1a pos3

1405.8
1249.6
1093.4
937.2
781
624.8
468.6
312.4

211.5

156.2

0
180
Cytb

160

140

120
CoI

100

80

60
NaK

40

20

0
Ef1a

1562

10-3 Net Phylogenetic Informativeness

1480.5

160

Cytb pos12

1903.5
1692

1249.6

10-3 Net Phylogenetic Informativeness

10-3 Net Phylogenetic Informativeness

Heterarthrinae

180

1405.8

S066 Sco vic2

6i Sco bet1
S031 Sco bet2
S032 Sco vic1
D5 Sco bet3
2k Sco tir2
1k Sco tir1
S075 Sco sp
5j Fla sp
S034 Fla gla
S033 Fla hor
3i Fla sep
1i Fla wue
S036 Fla nan2
6j Fla nan1
S073 Pro jap
3j Fnu pum1
S076 Fnu pum3
S037 Fnu pum2
S038 Fnu doh2
2j Fnu doh1
1j Fne nig1
S065 Fne nig2
5i Fne sp
4j Fnu ulm
9j Par res
Xr Par kam
8i Par ten
2i Hin exc1
S071 Hin exc2
9i Hin rec
4k Pro tho
Xj Pro pyg
S064 Pro luc
S021 Het ace
S061 Het cun
8k Het hea
S077 Het imb
9k Het leu
P7 Het nem2
5k Het nem1
S022 Het vag2
6k Het vag1
S024 Het mic3
S025 Het mic4
S023 Het mic2
7k Het mic1
Xk Het och
8j Met pum
S035 Met sp
7j Met lan1
S063 Met lan2
S074 Met roh
S062 Met alb2
4i Met alb1
3k Not sur
Xi Not sp
S082 Sil lho
P4 Phy ate
S039 Hop xyl
S080 Per sp
N8 Ard bru
S026 Ble phy
MX End aet
S030 Pac rap
L1 Ten not
S040 Sio ruf
S069 Emp fle
N7 Cal sp
P8 Str tac
S079 Nes mor
E4 Eit gre
AX Pon pus
A5 Ama amp
E5 Sta com
K2 Pla lur
J2 Pse fus
JX Cla com
H8 Sus ann
N9 Eri ova
D2 Ath cir
S090 Tri sp
L2 Abi can
L4 Dip sim
M8 Ste sp
S086 Arg sp
S084 Lop ana
S085 Bla fil

0
180

160

140

120

100

80

60

40

20

Cytb pos1

CoI pos1

NaK pos1

EF1a pos1

Cytb pos2

CoI pos2

NaK pos2

EF1a pos2

Cytb pos3

CoI pos3

NaK pos3

EF1a pos3

Fig. 3. Net phylogenetic informativeness curves for the different partitions of the sequence data in relation to time and the relaxed molecular-clock tree (A). Phylogenetic
informativeness curves are shown for each gene (B), for codon positions 1 + 2 and 3 within each gene (C), and for all three codon positions within each gene (D). See legends
below plots for curve colors for each partition.

leaf-mining in the Tenthredinidae is identied within the distantly

related subfamily Nematinae (Fig. 2A), where the leaf-mining tribe
Pseudodineurini is represented in this study by Pseudodineura fuscula (Klug) (see also Nyman et al., 2006b).
Host-plant use in Heterarthrinae is versatile: The included species can be found on 15 different plant genera, but many of these
are found in several locations in the miner phylogeny (Fig. 2A).
Despite the frequent host shifting, the tendency of closely related
miner species to share host genera leads to statistically signicant
phylogenetic conservatism as assessed by the PTP test (P < 0.001)
and the likelihood-ratio test of Pagels lambda (estimated k = 1,
ln L = 82.72; xed k = 0, ln L = 102.32; v21 39:21, P < 0.001).
Nevertheless, the maximum-likelihood reconstruction of ancestral
host associations rapidly becomes uncertain towards the base of
the ingroup (Supplementary Fig. 5A). The uncertainty is not particularly affected by taking phylogenetic uncertainty into account by
tracing host genera on 1000 post-burnin trees (Supplementary
Fig. 5B).
The topology of the plant phylogeny differs strikingly from that
of the leaf-miners (Supplementary Fig. 6). According to the numerous pareto-optimal reconstructions found by TreeMap, explaining
extant minerplant associations requires a maximum of 28

codivergence events (in addition to a variable but large number

of duplications, switches, and losses), which does not exceed the
expectation based on randomizing the miner tree 100 times
(P = 0.080, 95% CI 0.0280.141). More importantly, estimated
divergence times among heterarthrine lineages are in most cases
far younger than corresponding splits in the plant tree, meaning
that 95% highest posterior density intervals of node ages in most
cases do not cross the expectation of equal ages (Fig. 4). On the
other hand, eight relatively recent nodes on the sawy tree are
roughly contemporaneous or even older than the corresponding
splits in the plant tree, but the interpretation of most of these is affected by the ancestral state reconstructions at the nodes (see
below).

4. Discussion
4.1. Origin and phylogeny of heterarthrine leaf-miners
Our sequence-based phylogenetic analyses of heterarthrine leafminers and representative outgroup taxa from the Tenthredinoidea
produced trees that are generally strongly supported, particularly

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S.A. Leppnen et al. / Molecular Phylogenetics and Evolution 64 (2012) 331341

160
140

100
80
20

60
5

Miner node age (My)

120

19

13

40
16

12

20

18

0
0

20

40

60

80

100

120

140

160

Plant node age (My)

Fig. 4. Comparison of estimated ages of corresponding nodes in the phylogenies of
heterarthrine sawies and their host plants. Numbers adjacent to some of the
points refer to specic nodes in Fig. 2A that are discussed in the text; error bars
show 95% condence intervals for plant and miner node ages. The dashed line
shows the expectation (x = y) if lineage splits in the miners and plants would have
occurred at the same time.

for groupings within Heterarthrinae (Figs. 1 and 2A). Bayesian and

ML trees are largely congruent, while MP results present some clear
contradictions to the model-based analyses. However, all of the
apparently conicting groupings in the MP trees receive very weak
bootstrap support (Supplementary Fig. 2), which points to the conclusion that model-based analytical methods simply outperform
maximum parsimony when resolving the older divergences of the
tree. Therefore, we base our inferences below mainly on the Bayesian and ML trees.
The comparatively good resolution achieved within the Heterarthrinae apparently results from an appropriate level of phylogenetic informativeness in the sampled genes across the focal time
period (Fig. 3). PI in the data appears to arise mainly from the third
codon positions of the two mitochondrial genes and the nuclear
NaK gene, while the slower-changing EF-1a gene contributes less
information. On the other hand, it must be noted that the comparatively high net PI of NaK partly follows from the fact that the sequenced portion of NaK is longer than those of the other genes
(Table 2), meaning that relative per-site PI of NaK would be slightly
lower. Based on the MCC chronogram and the PI curves, the applied set of genes should prove useful also for resolving relationships within other tenthredinid subfamilies. By contrast, the
parallel dips in net PI estimates towards the past in combination
with the short internodes in the backbone parts of Tenthredinidae
(Fig. 2) means that additional data will be needed for elucidating
branching patterns among tenthredinid subfamilies.
The phylogenies reveal that larval lifestyles have shifted from
external feeding on plants to various internal feeding modes
(leaf-mining, gall-making, shoot-boring, and leaf-rolling) several
times within the Tenthredinoidea, but particularly within its most
diverse family, Tenthredinidae. More importantly, the phylogenetic
trees bring order to the composition of the subfamily Heterarthrinae, which has been debated for decades (cf. Goulet, 1992;
Pschorn-Walcher and Altenhofer, 1989; Smith, 1971; Taeger et al.,
2010): As currently dened, Heterarthrinae is clearly polyphyletic,
because leaf-skeletonizing Caliroa and Endelomyia are related
neither to leaf-miners, nor to each other. Thus, they should be included within Blennocampinae rather than in Heterarthrinae, with
the caveat that, like many morphologically dened subfamilies of

Tenthredinidae, blennocampines seem to constitute a heterogeneous taxon.

According to our results, heterarthrine leaf-miners constitute a
monophyletic clade that originated from external-feeding blennocampine ancestors c. 11080 million years ago. However, additional sequence data and increased taxon sampling will be
needed to pinpoint the exact phylogenetic position of the South
American Nothofagus-associated genus Notofenusa, as well as the
Mediterranean Phillyrea-mining Silliana lhommei (Figs. 1 and 2A).
At present, it seems safest to retain these within Heterarthrinae,
in accordance with the currently prevailing view that transitions
from external feeding to leaf-mining are rare in plant-feeding insects (Hartsough et al., 2007; Sinclair and Hughes, 2010). Within
Tenthredinidae, such shifts probably have occurred only twice,
the other origin being the small, mostly Ranunculaceae-mining
tribe Pseudodineurini in the subfamily Nematinae (Fig. 2A and
Supplementary Fig. 4; see also Nyman et al., 2006b).
The phylogenetic analyses also shed light on the species status
of some problematic leaf-miners. Scolioneura betuleti and S. vicina
have been regarded as different species on the basis of phenological differences: S. vicina has been considered an early-season species, while S. betuleti should feed later, in the autumn (Altenhofer
and Taeger, 1998). However, the fact that individuals representing
these presumed species are intermingled with each other on the
tree supports MacQuarrie et al.s (2007) proposal to synonymize
S. vicina with S. betuleti. Likewise, the shallow genetic divergences
found among four Acer-feeding Heterarthus lineages indicate that
their species status should be conrmed by molecular methods.
4.2. Evolution of host-plant use
While individual heterarthrine species typically are conned to
utilizing one or a few congeneric plant species (Altenhofer, 2003;
Smith, 1979; Taeger et al., 1998) and closely related miners tend
to share host genera, our phylogenetic analyses uncover considerable evolutionary lability in species-level host-plant use over longer time scales (Fig. 2A and Supplementary Fig. 5). As a
consequence, the phylogenies of the miners and their hosts are
strikingly different, and splits among heterarthrine lineages are
for the most part far younger than corresponding nodes on the
plant phylogeny (Fig. 4). This kind of a comparison is relatively
crude, especially when it comes to older miner clades that encompass multiple host taxa, and molecular dating is still an imprecise
endeavor (Battistuzzi et al., 2010; Forest, 2009; Magalln, 2010),
but the clearly non-overlapping 95% highest posterior density
intervals of most miner vs. plant nodes nevertheless point to the
conclusion that methodological errors are an unlikely cause for
the interpretations. For example, the plant lineages leading to Salicaceae and Betulaceae diverged over 100 million years ago, but
three putative shifts among genera belonging to these families
have occurred in the Heterarthrinae within the last 50 million
years (in Scolioneura, Fenusella, and Heterarthrus; Fig. 2A).
Out of the 24 selected miner-phylogeny nodes, only eight relatively recent ones are roughly contemporaneous with divergences
in the plant phylogeny (Fig. 4). Two of these involve potential trackings of the initial split between the salicaceous genera Populus
and Salix in Fenusella and Heterarthrus (nodes 2 and 16 in
Fig. 2A). The interpretation of contrasts at nodes 1820, and 12
13 is more complicated: In both cases, only a single shift is required, but its age depends on the ancestral-state reconstruction
at the focal nodes. In particular, the shift from Rubus to Geum within Metallus probably occurred at or after node 18, rather than at 19
or 20 (see Supplementary Fig. 4), which would make also this shift
younger than the split between the rosaceous host genera. The
only case in which a branching event in the sawy tree clearly predates the split of the hosts occurs at node 5, but this most likely

S.A. Leppnen et al. / Molecular Phylogenetics and Evolution 64 (2012) 331341

simply indicates that the shift between Alnus and Betula within Fenusa occurred sometimes after the divergence of the lineages leading to F. pumila and F. dohrnii. Overall, our results support a
growing body of evidence that diversication times of herbivorous
insects generally postdate those of their hosts, meaning that present host-use patterns in insects mainly result from a pattern of
host conservatism punctuated by occasional shifting among preexisting plant lineages (Gmez-Zurita et al., 2007; Hunt et al.,
2007; Lopez-Vaamonde et al., 2006; McKenna et al., 2009; Ohshima et al., 2010; Winkler et al., 2009; but see Kergoat et al., 2011).
The main source of conict between the phylogenies of heterarthrine leaf-miners and their host plants (Supplementary Fig. 6)
is that shifts have occurred repeatedly among a few dominant
northern plant genera (including Betula, Alnus, Salix, and Populus)
that are in many cases distantly related (Supplementary Fig. 3;
Burleigh et al., 2009; Wang et al., 2009). In this respect, the heterarthrine host repertoire and shifting pattern shows some remarkable similarities to those of other Holarctic insect herbivore taxa,
including nematine sawies (Nyman et al., 2006b, 2010), chrysomeline leaf beetles (Mardulyn et al., 2011; Termonia et al., 2001),
leaf-mining Phyllonorycter moths (Lopez-Vaamonde et al., 2003),
and several agromyzid y genera (Nyman et al., 2002; Scheffer
et al., 2007). Such convergent biases in shifting probabilities in
multiple distantly related insect groups most likely explain the fact
that the plant genera preferred by heterarthrines rank among those
supporting the highest overall diversities of insect species in Europe (Brndle and Brandl, 2001; Kennedy and Southwood, 1984).
Explaining the use of plant genera by the sampled 38 heterarthrine species with known hosts requires a minimum of 20 shifts
(maximum-parsimony steps) across the pruned MCC tree
(Supplementary Fig. 5). It must be noted that the true evolutionary
history of host use in Heterarthrinae must have been even more
complex, because representatives of several rare miner genera
could not be obtained for our study, and because the hosts of many
species-poor East Asian and South American taxa are still unknown
(Supplementary Table 1). Adding unsampled miner genera to the
phylogeny would add some divergent plant groups to the overall
host repertoire (e.g., Viola (Violaceae) used by Nefusa, and Platanus
(Platanaceae) used by Bidigitus), but would most likely also
uncover additional convergent host shifts (e.g., Anafenusa occurs
on Acer and Ulmus). Nevertheless, adding taxa would not change
the main interpretations above, i.e., that the phylogenies of heterarthrine leaf-miners and plants are highly discordant, and that
most of the observed shifts in host use occurred well after the
divergence of the plant groups involved.
The demonstration of frequent shifts by heterarthrines across
available plant taxa supports the view that diversication in herbivorous insects is to a large extent driven by specialization onto
alternative host plants (Ehrlich and Raven, 1964; Fordyce, 2010;
Slove and Janz, 2011; Winkler and Mitter, 2008). However, other
speciation modes most likely also operate in the group: The aforementioned Scolioneura betuleti + S. vicina pair may constitute a case
of allochronic (=phenology-driven) speciation (cf. Abbot and
Withgott, 2004; Santos et al., 2011), and non-ecological allopatric
diversication is implicated in several cases in which closely related species (e.g., within Fenusella, Parna, and Heterarthrus) have
identical or broadly overlapping host-plant ranges. In addition,
identifying the underlying cause is difcult in the cases in which
sister species differ in both host-plant use and geographic range
(e.g., the Central European Fenusella wuestneii feeds on Salix viminalis while its Northern European sister species Fenusella septentrionalis is apparently a specialist on Salix lapponum, and Asian Parna
species attack different Tilia species than do the European ones)
(Supplementary Table 2). The heterarthrine leaf-miners evidently
constitute a promising model system for studying the relative
importance of different speciation modes in herbivorous insects,

339

but such analyses will require denser taxon sampling also within
the already-included genera, as well as more detailed information
on the geographical distributions and host-plant use of the sampled species (cf. Imada et al., 2011; Nyman et al., 2010).
5. Conclusions
Our phylogenetic analyses indicate that leaf-mining sawies in
the subfamily Heterarthrinae form a monophyletic clade, while
caliroine leaf-skeletonizers in Endelomyia and Caliroa are distantly
related to the leaf-miners; therefore, the current denition of Heterarthrinae should be revised to exclude the caliroine genera.
Although individual heterarthrine species are highly specialized
in their use of available plants, the phylogenetic trees of heterarthrines and their hosts are discordant, and many cladogenetic events
in the leaf-miner tree clearly postdate corresponding splits in the
plant phylogeny. Current host-plant associations within Heterarthrinae therefore evidently have been produced by a combination
of host conservatism and occasional shifts among plant lineages.
Many host-plant shifts have occurred onto plant genera that already harbor other heterarthrines. Similar convergent colonizations and back-colonizations among distantly related hosts have
been found also in several other herbivorous insect groups, and
may reect, for example, retention of ancestral feeding capabilities
after a shift (Janz and Nylin, 1998; Slove Davidson, 2012), shared
ecological traits among the host taxa (Mardulyn et al., 1997),
and/or host abundancedependence in the probability of successful establishment and long-term survival (Nyman, 2010). As in
many other herbivorous insect taxa, diversity within Heterarthrinae appears to have been generated through multiple mechanisms,
and possible cases of sympatric or allopatric ecological speciation,
allopatric non-ecological speciation, and allochronic speciation can
be identied based on our analyses; however, a more thoroughly
sampled phylogeny of the subfamily is needed for estimating the
relative proportions of different speciation modes within the
group.
Acknowledgments
We would like to thank colleagues who provided specimens for
this study: David R. Smith, Susanne S. Schulmeister, Rob Edmunds,
Veli Vikberg, Jukka Matero, Jean-Luc Boev, Klaus Hellrigl, Marko
Mutanen, Kazuo Yamazaki, Matti Viitasaari, Alexey G. Zinovjev,
Heikki Roininen, Herbert J. Jacobson, Mikk Heidemaa, Jeffrey B.
Joy, Jorma Sorjonen, and A. Ueda. We are especially grateful to
David R. Smith, who identied North American leaf-miner specimens. Michael Charleston, Renske Onstein, Richard Carter, and
Tobias Malm are thanked for helpful discussions concerning data
analyses. Funding for this project was provided by the Academy
of Finland (Projects 124695 and 14868 for TN).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.ympev.2012.
04.005.
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