Molecular phylogeny of Eois (Lepidoptera, Geometridae): evolution of wing patterns and host plant...

Molecular phylogeny of Eois (Lepidoptera, Geometridae): evolution of wing patterns and host plant use in a species-‐rich group of Neotropical moths Patrick Strutzenberger, Gunnar Brehm, Florian Bodner, Konrad Fiedler

Article first published online: 5 AUG 2010 DOI: 10.1111/j.1463-‐6409.2010.00440.x The definitive version is available at www.blackwell-‐synergy.com

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Strutzenberger, P., Brehm, G., Bodner, F. & Fiedler K. (2010). Molecular phylogeny of Eois

(Lepidoptera, Geometridae): evolution of wing patterns and host plant use in a species rich group

of Neotropical moths. Zoologica Scripta, 00, 000-000.

Eois is a pantropical genus of Geometridae moths with currently 250 valid described species, the

majority of which occur in the Neotropics. Eois is a prominent component of Andean moth

communities locally accounting for up to ~10% of geometrid individuals. We address the

evolution of wing patterns and host plant use in Neotropical Eois and provide a preliminary

assessment on the monophyly and biogeographic history of the entire genus as well as affinities

within the subfamily Larentiinae. We applied Bayesian, maximum likelihood and maximum

parsimony methods of phylogenetic reconstruction to a 142 taxon dataset of partial COI (1220bp)

and Ef1α (1066bp) sequences resulting in the largest taxon set of geometrid moths analyzed in a

molecular phylogenetic study so far. Monophyly of Eois was always strongly supported. Ten

monophyletic clades were found with good support, seven of which have characteristic wing

pattern phenotypes. Only one wing pattern type occurs in two clades. Trophic associations with

representatives of the family Piperaceae occur in all 8 (of 9) Neotropical clades for which host

information is available. Apart from feeding on Piper, at least two Eois species in Ecuador feed

on Peperomia, and one on Manekia (all Piperaceae); two further species live on Hedyosmum

(Chloranthaceae). Species feeding on Peperomia, Manekia and Hedyosmum are usually nested in

Piper-associated clades. Single records of associations with Gesneriaceae and Monimiaceae are

scattered in otherwise Piperaceae-associated clades. These patterns suggest multiple parallel host

shifts away from Piper as ancestral food plant. Old World Eois were recovered as monophylum

and sister to Neotropical Eois. Within the subfamily Larentiinae the genus Eois has previously

been placed close to the tribe Eupitheciini, but this was not supported in our phylogenetic

analyses.

Patrick Strutzenberger, Department of Animal Biodiversity, University of Vienna, Rennweg 14,

1030 Wien, Austria. E-Mail: [email protected]

Gunnar Brehm, Institut für Spezielle Zoologie und Evolutionsbiologie mit Phyletischem Museum,

Friedrich-Schiller-Universität Jena, Erbertstraße 1, 07743 Jena, Germany. E-Mail:

[email protected]

Florian Bodner, Department of Animal Biodiversity, University of Vienna, Rennweg 14, 1030

Wien, Austria. E-Mail: [email protected]

Konrad Fiedler, Department of Animal Biodiversity, University of Vienna, Rennweg 14, 1030

Wien, Austria. E-Mail: [email protected]

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1. Introduction

Eois is a speciose genus of rather small tropical geometrid moths assigned to the subfamily

Larentiinae. Scoble (1999) recognized 250 valid species; only two further species were described

since then (Herbulot 2000). The genus is distributed in the Americas, ranging from Mexico to

Argentina, as well as in southern Asia (21 species), Australia/Oceania (9 species) and Africa (15

species). The large majority of described species (205) occur in the Neotropical region where

Eois comprises an important part of megadiverse geometrid moth assemblages. For example, 60

morphospecies of Eois were sampled along a continuously forested elevational gradient in central

Costa Rica (40–2730 m a.s.l.), which accounted for 8.1% of the geometrid species and 7.8% of

geometrid individuals in that study (Brehm et al. 2007). Tropical Andean forests harbour the

globally highest diversity of geometrid moths, including Eois. Over the past 10 years, the

geometrid fauna of one particular area in southern Ecuador, the Reserva Biológica San Francisco

(RBSF) and adjacent areas (1000–2700 m a.s.l), has served as a paradigm to investigate patterns

in species diversity and community structure of tropical moths (e.g. Brehm et al. 2003a, b, 2005;

Brehm & Fiedler 2003, 2005; Fiedler et al. 2008). Brehm et al. (2005) recorded 102 Eois species

in the area, representing 8.1% of geometrid species and 10.2% of all geometrid individuals.

However, recent DNA barcoding data and additional field sampling suggest that the true number

of Eois species in the RBSF area alone may easily exceed 160 species (Strutzenberger et al. 2010).

Hence, the overall species count of Eois is likely to be considerably higher than 250, perhaps

being twice or three times as high globally. The phylogenetic history of Eois has so far never

been addressed. This is surprising since close co-evolution with host plants has been postulated

by Dyer et al. (2004) and Connahs et al. (2009). Moreover, Eois species are very diverse in terms

of wing patterns and coloration, suggestive of a striking case of radiation. Eois has circumtropical

distribution; still the monophyly of the genus or even just the Neotropical members has so far

never been tested. Life-history data for 95 morphospecies of Neotropical Eois has been gained in

recent years as part of massive campaigns to elucidate tropical food webs. Most published host

plant records for Neotropical Eois refer to Piper plants (Bodner et al. in press; Dyer & Gentry

2004; Dyer et al. 2009a, b; Janzen & Hallwachs 2009). Recently added records by Dyer et al.

(2009) and Strutzenberger (2009) show Eois to also feed on the piperacean genus Peperomia as

well as a handful of cases scattered over other families (Chloranthaceae, Gesneriaceae and

Monimiaceae; Table S1). However, it has not yet been tested in a cladistic framework how much

phylogenetic signal is present in host plant relationships of Eois.

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The specific aims of the present study are: (1) to assess the phylogenetic distribution of host plant

affiliations in Neotropical Eois, especially with Piperaceae, (2) to search for phylogenetic

substructure within Neotropical Eois in relation to wing patterns, (3) provide the basis for a

forthcoming species level taxonomic revision of Neotropical Eois, (4) to assess the monophyly of,

and provide a first phylogenetic hypothesis for, the morphologically diverse genus, and (5) gain

first insight into the relationship of Eois to other larentiine genera . Apart from Canfield et al.

(2008) this is one of the first studies to investigate evolutionary dynamics in a diverse clade of

geometrid moths with dense taxon sampling and down to the species level. Hence, results of such

a case study promise to yield insights into the historical dimension of species diversity in one of

the most species-rich insect families.

2. Material and methods

Collection and DNA sequencing

Most Eois moths used in this study were collected in 2005 within the Reserva Biológica San

Francisco in southern Ecuador in an area of ca. 2.5 km2 (03°58.5’–3°59.7’S, 79° 04.1’–

79°05.1’W, 1800–2700 m a.s.l.). Additional sampling was carried out in November 2008 in

Podocarpus National Park at the Bombuscaro entrance (04°06.9’S, 78°58.1’W, 1020 m a.s.l.).

Old world taxa were collected at Mt. Kinabalu, Borneo, Malaysia, and in the Oribi Gorge Nature

Reserve, South Africa. Collection sites for all specimens used in this study are indicated in Table

1, for details about collection sites see Brehm & Fiedler (2003), Zimmermann (2005) and Günter

et al. (2008). Some representatives of possible outgroups were collected at sites in Austria, and

further published sequence data were used for outgroups where available (Table 1). Moths were

killed with cyanide, and stored at -20°C until extraction. Age of extracted specimens ranged from

2 months to 10 years. Pre-PCR and post-PCR work was separated by performing the relevant

tasks in separate rooms and the use of a PCR workbench with UV decontamination. Heads and/or

legs of moths were homogenized with ceramic beads using a Precellys 24 homogenizer set to

5000 min-1 for 2x20 s. DNA extraction was performed with the DNEasy Tissue Kit (Qiagen), the

Peqgold Tissue DNA mini Kit (Peqlab), or the Macherey and Nagel NucleoSpin Tissue kit

according to the protocol supplied with the respective kit. The target fragments were amplified

using the Fermentas PCR system. Target regions were the nuclear gene coding for translation

elongation factor 1-alpha (Ef1α) and the mitochondrial gene coding for cytochrome oxidase

subunit I (COI) (see Table S1 for primers). PCR reactions were set up with 2.5 µl of 10x

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(NH4)2SO4 PCR Buffer, 2 µl 25mM MgCl2, 0.1µl 10mM dNTPs, 1µl of each primer (10µM), 1µl

genomic DNA, 1 unit Taq Polymerase and filled to 25µl with PCR grade H2O. PCR reactions

were purified by digestion with Shrimp Alkaline Phosphatase and Exonuclease I for 15 min at

37°C followed by 15min at 80°C for enzyme deactivation. Sequencing reactions were set up with

1 µl ABI BigDye 3.1, 1µl primer, 1-3 µl template DNA and filled to 10 µl with PCR grade H2O

and sequenced on an ABI capillary sequencer. All gene fragments were sequenced in both

directions. PCR and sequencing primers as well as thermal cycler programs are indicated in Table

S1.

Identification of moths and examination of wing patterns

Photographs were taken either of the wings of the right body side or of spread moths in dorsal and

ventral view on an Olympus optical device or a Nikon D80 DSLR camera, processed and

examined using Adobe Photoshop versions 8 and 9 and Cell_D version 3.2. Moths were

identified by comparison of wing patterns with type material or photographs of type material

(98% of all described Neotropical species available). All identifications must be seen as

preliminary until formal revisionary work will be carried out, including examination of genitalia

morphology and molecular data of type specimens. Most of the species examined in this study are

still formally undescribed. In these cases, the assignment of names only provides an

approximation of the true identity of species. The assignment of names like 'E. spnr goodmanii' to

more than one taxon does not imply conspecificity of these taxa. It is merely a statement on what

we conclude is the closest described relative of that taxon.

Selection of taxa for phylogenetic analyses

We included 80 species of Eois clearly distinguishable as separate species by their wing pattern

morphology and another 20 Eois species identified by an integrative taxonomy approach

(Schlick-Steiner et al. 2010) where differences in COI barcode sequences could subsequently be

confirmed by consistent differences in wing pattern upon re-examination. Furthermore we

included 10 Eois taxa which are putative species defined by a >2% pairwise distance in COI

barcode sequences. Even though all but two of our Neotropical samples were collected on a small

geographic range in southern Ecuador, examination of 98% of all Neotropical Eois type

specimens preserved in natural history museum collections revealed that our dataset covers the

vast majority of the known morphological diversity in that moth genus. Only two rare (one

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species each) morphotypes present in type material are apparently distinct from all morphotypes

included in this study. Sampling of moth communities on an extensive elevational gradient (40-

2730m) in Costa Rica (Brehm et al. 2007) yielded no species of Eois that is obviously distinct

from all clades represented in our samples from Ecuador. Our sampling does therefore provide

representative, but not exhaustive, coverage of known Neotropical Eois. It cannot be excluded

that currently unknown morphotypes will be discovered when sampling effort is extended. Our

sampling of Old World Eois is much less complete in that respect. We examined material and

literature on Neotropical Geometridae for Eois species that may have erroneously been assigned

to other genera, but no such cases have been detected so far. On the contrary, some species

appear to be erroneously assigned to Eois (G. Brehm et al., unpublished data). None of those

species were available for sequencing.

Outgroup taxa were in part (21 taxa) taken from Genbank and partially acquired by ourselves (9

taxa). We used all sequences for Larentiinae and Sterrhinae available in Genbank provided that

both COI and Ef1α sequences were available for the same taxon. As the sister group of Eois is

unknown we used a broad selection of outgroup taxa to gain an initial assignment of Eois among

the major tribes of the Larentiinae. Outgroup taxa represent the tribes Cidariini, Cataclysmiini

Hydriomenini, Melanthiini, Perizomiini, Phileremini, Trichopterygini, Xanthorhoini as well as

the tribe Eupitheciini to which Eois has been assigned on morphological characters albeit without

the application of phylogenetic methods (Holloway 1997). Definition and relationships of tribes

within the Larentiinae is generally disputed and has mostly not been tested by phylogenetic

methods. We included the Sterrhinae next to the Larentiinae because members of those two

subfamilies are often very similar in their appearance. Misassignments are therefore easily

possible between those two subfamilies; see Ounap et al. (2008) for a recent example in the

comparatively well-known European fauna. In order to advance our knowledge on the placement

of Eois within the Larentiinae it is vital to use outgroup taxa with a well established taxonomy

which is not the case for the large majority of small sized geometrid taxa found in Ecuador. For

that reason most outgroup taxa are Palaearctic species. All trees were rooted with Archiearis

parthenias, which has been found to lie outside of the clade (Larentiinae + Sterrhinae) in

molecular as well as morphological analyses (Yamamoto & Sota 2007; Young 2008) and is

therefore well suited as outgroup for the present analysis. The inclusion of more distant outgroups

(e.g. Ennominae, Geometrinae) produced erratic placements (P. Strutzenberger unpublished

results) presumably due to lack of phylogenetic signal. Taxa were included only when at least 3

of the 4 PCR fragments could be successfully sequenced, ensuring that at least one half of each

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gene was available for phylogenetic analysis. The same quality standard was applied to sequences

taken from Genbank. Chimeric taxa were created when sequences of COI and Ef1α could not be

obtained from the same individual. In these cases the Ef1α sequence was taken from a different

individual belonging to the same species with identical COI barcode sequence.

Sequence data processing

Proofreading of sequences and contig assembly was done with ChromasLite Version 2.01,

ChromasPro Ver 1.34 and DNAStar Lasergene SeqMan Pro Ver. 7.1 and 8. Heterozygous

positions were coded as ambiguities. Assembled sequences had a length of 1220 bp and 1066 bp

for COI and Ef1α, respectively. All sequences were aligned manually using Bioedit Ver 7.0.4.1

(Hall 1999). Sequence data were prepared for analyses using the programs FORCON version 1.0

and MEGA version 4 (Kumar et al. 2004; Tamura et al. 2007). Sequences were screened for

unusual nucleotide composition and the presence of erratic stop codons to control for possible

pseudogene amplification. Transition/transversion ratios, pairwise distances and nucleotide

frequencies were calculated using PAUP* 4b10 (Swofford 1999) and imported into OpenOffice

3.1.0 for analysis and graphic display. To assess possible incongruence of the two genes a

partition homogeneity (incongruence length difference) test was performed with PAUP.

Parameters were 1000 replicates of heuristic search, TBR branch-swapping, random addition

sequence and a time limit of 180 s per replicate to limit the otherwise excessive computation time.

Selection of partitioning strategy for Bayesian and maximum likelihood analyses

Partitioning of sequence data is a major factor influencing results of maximum likelihood and

Bayesian analyses of phylogeny (e.g. Brandley et al. 2005; McGuire et al. 2007). We therefore

applied objective criteria to select the preferred partitioning scheme for our dataset. For this

purpose we compared five different strategies (Table S3) using the Akaike Information Criterion

(AIC) and Bayes factors. AIC values were calculated from the likelihood of the best tree for each

partitioning regime obtained during search for the best known likelihood tree with RAxML

(Stamatakis 2006a; Ott et al. 2007) using the GTRMIX model where trees are calculated using

the computationally more efficient GTRCAT model (Stamatakis 2006b) and scored under

GTRGAMMA to allow for comparison of likelihoods. Search parameters were 100 replicates

with initial rearrangement settings determined automatically and 30 rate categories. Other settings

for both of the latter parameters were tested empirically but were not found to increase likelihood

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values. The number of parameters for calculation of AIC values was assumed as 9 parameters of

the GTR+G substitution model plus the number of branch lengths (calculated as 2n-3, with n

being the number of taxa). Selection of the preferred partitioning for Bayesian analyses was done

using the Bayes factor (Kass & Raftery 1995). Analyses were performed with the MPI version of

MrBayes 3.1.2. (Huelsenbeck et al. 2001; Ronquist & Huelsenbeck 2003; Altekar et al. 2004).

The best fitting substitution model for each partition was selected using MrModeltest 2.2 (Posada

& Crandall 1998; Nylander 2004) following the AIC and applied accordingly. Searches were

done for at least 5x10^6 generations or until the standard deviation of split frequencies dropped

below 0.01. Stationarity was evaluated by visual examination of a plot of –lnL versus the number

of generations. The first 10% of samples were subsequently discarded as burn-in in all analyses.

Bayes factors were calculated as described in the MrBayes 3.1.2 manual based on the harmonic

mean of marginal likelihoods obtained from the sump command. The preferred partitioning

strategy was subsequently applied in all Bayesian and maximum likelihood analyses.

Phylogenetic analysis

Bayesian inference of phylogeny was done with the MPI version of MrBayes 3.1.2. The preferred

partitioning regime selected by Bayes factors as described in the previous section was applied.

Analyses were run for 10^7 generations with a sample frequency of 100. Chain temperature was

set to 0.1 in order to achieve a proportion of successful chain swaps between 0.1 and 0.7. The

burn-in was determined by visual examination of a plot of –lnL against the number of generations

and was set to 10000, corresponding to the first 10^6 generations, for all analyses.

Maximum likelihood analyses were performed with RAxML 7.0.4. The dataset was partitioned

according to the preferred strategy selected by the AIC. The GTR+G substitution model was

applied to all partitions as RAxML, the author of RAxML argues against the use of the GTR+G+I

model stating that the number of invariant sites and gamma cannot be optimized at the same time.

A search for the best known likelihood tree was performed with 200 replicates, automatic

determination of rearrangement settings and 30 rate categories. Bootstrapping was done using the

rapid bootstrapping algorithm implemented in RAxML with 10000 replicates with remaining

parameters and partitioning as stated above. Bootstrap support values were then drawn onto the

best known likelihood tree. Maximum parsimony analyses were performed with PAUP* 4b10 set

to 1000 replicates of heuristic search with tree-bisection-and-reconnection branch swapping and

random addition sequence. In the case of separate analyses of Ef1α sequences a time limit of 180

seconds was imposed for each of the 1000 replicates to prevent excessive and nonsensical

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swapping of nodes that cannot be resolved by Ef1α sequences. Bootstrapping (Felsenstein 1985)

was done with PAUP* 4b10 using the bootstrap command set to 1000 replicates of 10 heuristic

search replicates each, heuristic search parameters were tree-bisection-and-reconnection branch

swapping and random addition sequence. For bootstrapping of Ef1α trees a time limit of 90

seconds per heuristic search replicate was imposed for reasons stated above. Uninformative

characters were excluded from all parsimony analyses. Strict consensus trees were calculated

from the obtained most parsimonious trees using PAUP* 4b10, bootstrap support values were

manually added to the corresponding nodes. Trees were examined and edited with MEGA version

4, Figtree version 1.1.2 and Adobe Illustrator version 14.

Host plant records

Host plant records for Eois were in part acquired by ourselves (see Bodner et al. (in press) for

methods) or were collated from Bodner et al. (2010), Dyer et al. (2009), Dyer & Gentry (2009),

and Janzen & Hallwachs (2009). Due to the poor state of taxonomy of Eois moths, host plant

records were only used when a picture of the corresponding adult moth was available to us since

reliable assignment of species names is otherwise not possible in most cases. Records from

Janzen & Hallwachs (2009) were gathered by creating a list of host plant records using the option

'List of food plant records' to search for 'Eois'; the resulting table was then exported into a

spreadsheet where all records lacking an image of the adult in the 'Adult photographs' section of

the database were eliminated. The remaining specimens were identified by the image contained in

the 'Adult photographs' section, which lead to the split of the caterpillar species 'Eois Janzen01'

into two adult morphospecies. The species we split from Eois Janzen01 was informally termed

'Eois Janzen01_1'. Host plant records taken from online databases and literature are summarized

in Table S1 together with our novel records presented in this study. Maximum parsimony

optimization of host plant records onto the tree topology gained from total evidence Bayesian

analyses was performed using MacClade version 4.08 (Maddison & Maddison 2003). In parallel

we used Mesquite (Maddison & Maddison 2009) to obtain a maximum likelihood reconstruction

of ancestral states using the same character matrix. In order to allow the use of as much of the

sparse host plant information as possible, records for species within species complexes, where

morphological determination was impossible or at best doubtful, were treated as records for the

entire species complex. Records not assignable to a morphospecies represented in our dataset

were assigned to a closely related morphospecies which then serves as a proxy for the missing

species, when possible. These assignments were also made by wing pattern morphology, and

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were only accepted in cases where we are confident that the species for which the host plant

record is available is likely to be the sister species or belongs to the same species complex. In the

case of Old World Eois where no more specific data was available host plant records were

assigned on clade level (see Megens et al. 2005, for a similar approach). In this case the entire

Old World clade serves as proxy for the two known host plant associations for Old world Eois.

The association of Old World Eois with invasive Piper plants was not coded in our data matrix.

The applied character coding is given in the legend of Figure S5 and Eois taxa used are indicated

in Table S1.

3. Results

Sequence statistics

We acquired COI and Ef1α sequences for 121 taxa, 112 of which were of Eois. Of the 112 Eois

specimens 104 were collected in Ecuador, 5 in Borneo, 2 in Costa Rica and 1 in South Africa.

With the addition of sequences from 21 outgroup taxa obtained from Genbank our complete

dataset included 142 geometrid taxa. In 9 cases either COI or Ef1α sequences were of less than

90% the desired length. Chimeric taxa were created in three instances as indicated in Table 1.

Exclusion of taxa with incomplete or chimeric sequences had no effect on the relationships

recovered for the remaining taxa and no discernible effect on support values in any of the three

methods of phylogenetic reconstruction (results not shown). Sequenced specimens are listed in

Table 1. The sequence alignment of the COI gene has a length of 1536bp and the one for the

Ef1α gene 1225bp, amounting to a combined dataset of 2761bp. Alignment was straightforward

in all cases, no insertions or deletions could be detected, except for COI sequences of the two

Timandra species being shortened by one codon as reported by Ounap et al. (2008). A summary

of the number of characters and variability for each partition is given in Table 4. To asses the

potential for mutational saturation, transition/transversion ratios were plotted against pairwise

distances for first and second positions and separately for third codon positions (Fig. S1). It is

evident that the third positions of the COI gene become saturated with increasing pairwise

distances as transition/transversion (Ti/Tv) ratios approach 1 very soon. No saturation is present

in the Ef1α gene with Ti/Tv ratios almost exclusively above 1. COI sequences are AT-rich with

an average AT content of 71.1% whereas Ef1α sequences show a more balanced nucleotide

composition typical for nuclear genes with an average AT content of 48.1%. The average

uncorrected pairwise distance between COI sequences within Eois is 8.7% with a maximum of

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16.4%. The average distance between Ef1α sequences within Eois is 6.6% and the maximum is

13.5%.


Both employed measures favored partitioning of the dataset into 6 partitions corresponding to the

codon positions of both genes (Table 3). The preferred model has an Akaike weight of 1 and a

difference in Bayes factor of 351.14 when compared to the second best fitting model; this clearly

indicates that all other tested partitioning strategies are an inadequate approximation of the

sequence data. Substitution models selected by MrModeltest 2.2 are given in Table 3 for each

partition.

Combined Bayesian analyses resulted in a 50% majority rule consensus tree with an average

likelihood of –43863.973 (Figs. 1 and 2). Single gene Bayesian analyses resulted in consensus

trees with an average likelihood of –14782.601 and –28841.435 for Ef1α (Fig. S2) and COI (Fig.

S3), respectively. The Bayesian tree based on total evidence is shown in Figure 1 (Eois) and

Figure 2 (outgroup) with maximum parsimony bootstrap (mpb) and maximum likelihood

bootstrap (mlb) values added when ≥50. Maximum likelihood analyses of the combined dataset

yielded a best tree with a likelihood of –48015.943 (Fig. S1a). Analyses of the COI gene resulted

in a best tree with a likelihood of –31216.838 and analysis of the Ef1α gene resulted in a best tree

with a likelihood of –16035.796 (data not shown). Likelihoods of trees sampled during 200

replicates based on the COI gene alone were considerably more unstable than likelihoods

obtained from analyses of Ef1α and the combined dataset. Maximum parsimony analyses of the

combined dataset resulted in 26 most parsimonious trees (MPTs) with a length of 10586 steps

(CI=0.146, RI=0.502) (Fig. S1b). Analyses of the COI gene resulted in 168 MPTs with a length

of 7320 steps (CI=0.137, RI=0.394) and separate analysis of Ef1α resulted in 520421 MPTs with

a length of 3131 steps (CI=0.210, RI=0.675) (data not shown). The partition homogeneity test

resulted in p=0.001 which attests a significant incongruence between the COI and Ef1α gene.

Phylogenetic relationships within Eois

Trees obtained from combined analyses and single gene analyses of the Ef1α gene (Fig. S2) are

well resolved whereas trees obtained from COI sequences (Fig. S3) are only poorly resolved and

lack any meaningful support in deeper nodes. Only 2 of the 10 clades recognized within Eois

could be recovered as monophyletic in trees calculated from COI sequences. Trees calculated

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from Ef1α sequences alone are in most cases almost entirely congruent with the topology of the

combined analysis applying the same method of phylogenetic reconstruction except for some

young nodes that can be resolved by COI sequences. Incongruence between COI and Ef1α gene

trees is limited to deeper divergences. Subsequent presentation of results is therefore largely

based on trees calculated from the combined dataset, and special reference will be made when

referring to trees based on COI or Ef1α alone.

Eois is consistently recovered as monophyletic with full or near full support. Old World Eois are

consistently recovered as monophyletic receiving good support. The monophyly of Neotropical

Eois is not unequivocally supported (bpp=0.99; mpb=64). ML analyses show no support for

monophyly or non-monophyly of Neotropical Eois. All methods of phylogenetic reconstruction

agree in the recovery of 10 clades within Eois. All clades (except the ‘Old World clade’) are

named in Figure 1 after the oldest available species name contained in the respective clade. Most

of these clades share clear similarities with respect to wing pattern phenotypes, whereas others are

morphologically heterogeneous. The naming of the clades does not suggest any formal taxonomic

ranking such as subgenera.

With the exception of the adimaria clade the internal topology of these clades is reasonably well

supported and consistent among all three methods of phylogenetic reconstruction. The adimaria

and chasca clades are consistently associated with each other, as are the chrysocraspedata and

olivacea clades. Relationships of the clades relative to each other apart from the sister clade

associations mentioned below are in most cases only weakly supported and variable between the

different methods of phylogenetic reconstruction. There is good support for the association of the

adimaria clade, chasca clade, chrysocraspedata clade and the olivacea clade plus four taxa not

assigned to a clade with variable position. Placements of the odatis clade, trillista clade and

isographata clade within Eois are variable and not well supported. The catana clade is sister to all

other Neotropical Eois in Bayesian and MP analyses while ML analyses recover the clade as

sister to the Old World clade albeit without support. Within the Old World clade the single

African species we were able to include is firmly associated with a species from Borneo and

nested within other Asian species. No phylogenetic pattern of morphological features can be

recognized among Old World Eois, which is likely due to our small sample size.

Outgroup relationships

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MP and ML analyses recover Eois as sister to all other included Larentiinae except for Dyspteris

abortivaria, which is the first branching taxon in the Larentiinae. Bayesian analyses recover

Philereme transversata as putative sister to Eois, this placement is also recovered in single gene

Bayesian and ML analyses of the Ef1α gene. Relationships among outgroup taxa generally have

weak support. The Sterrhinae were recovered as monophyletic in Bayesian and ML analyses and

as paraphyletic in MP analyses. The topology within Sterrhinae is well resolved. D. abortivaria

was notably recovered either as sister taxon of all other Larentiinae (Bayesian and ML) or even as

the first branch after the root in MP analyses. To exclude potential negative effects of Dyspteris

abortivaria on the overall phylogeny we repeated combined analyses with all three methods of

phylogenetic reconstruction with D. abortivaria excluded. The resulting topology was identical to

trees including D. abortivaria and no change in support values were detected.

Host plant records

New host plant records were gathered in southern Ecuador for 35 species of Eois. Most of these

came from 11 different species (‘morphotypes’) of the plant genus Piper. Notable new host plant

records include Peperomia, with caterpillars being assigned to two different species in the E.

ciocolatina species complex by DNA barcodes (larval voucher codes: EoC00012 and EoC00013,

Genbank acc. no.: GQ433620 and GQ433621 respectively). One larva found on Hedyosmum sp.

was assigned to E. borrata Eo00518 by DNA barcodes (larval voucher code: EoC00014,

Genbank acc. no.: GQ433622). Two more species were also found to feed on a species of

Hedyosmum and were successfully reared and identified as E. spnr azafranata and E. spnr

golosata respectively. Furthermore, E. spnr restrictata was recorded from a species of Manekia

(=Sarcorhachis, Arias et al. 2006) (Piperaceae). For a complete list of host plant records see

Table S1. Combined with data from other sources 109 host plant records for approximately 100

species were available. 47 records could be matched to a morphospecies in our phylogeny, in 34

cases through conclusive similarity in wing pattern morphology; in 6 cases by DNA barcodes and

4 records were assigned to a closely related species. The three available records for Old World

Eois were assigned to the entire Old World clade as more specific data was not available.

Mapping of host plant records onto the phylogenetic tree (Fig. 1) shows that records are available

for all but one clade of Neotropical Eois (sagittaria clade). Maximum parsimony optimization of

host plant use reveals feeding on Piperaceae as the ancestral state for Neotropical Eois. Feeding

on Hedyosmum occurs at least twice in the adimaria clade. Feeding on Peperomia to our

14

knowledge occurs in the E. ciocolatina species complex in the odatis clade, and independently in

E. chasca, which is part of the chasca clade. E. chasca was also recorded on a species of

Alloplectus (Gesneriaceae) by Dyer et al. (2009a, b). Feeding on Monimiaceae is so far an

isolated occurrence, in the chrysocraspedata clade. The single species found on Manekia was

recovered in an early branching clade of Neotropical Eois. For the full maximum parsimony

reconstruction of ancestral states see Figure S5. Maximum likelihood reconstruction of ancestral

states (Figure S6) is in agreement with states reconstructed by maximum parsimony. The

maximum likelihood analysis provided additional information in two instances. First, Piper

feeding was recovered as the most likely ancestral state for the (adimaria+chasca) clade and

second, the ancestral host plant family for most of the adimaria clade was most likely the

Chloranthaceae.

4. Discussion


Frequently the partitioning of genes according to their codon positions is the preferred strategy

for Bayesian and maximum likelihood analyses (Brandley et al. 2005; McGuire et al. 2007). This

is also true for our dataset as both the AIC and the Bayes factor unequivocally selected this

particular partitioning strategy over any of the tested alternative strategies. Trees calculated from

COI gene sequences are poorly resolved except for young divergences. Analysis of the COI gene

was troubled by the high degree of homoplasy as expected from the Ti/Tv ratio plot. The

contribution of the COI gene to the basal branching pattern is therefore limited. The COI gene’s

lack of resolution for basal branchings is also likely to be the reason for the observed unstable

likelihood values during ML tree searches compared to the Ef1α gene. Major shifts in tree

topology cause noticeable variation of likelihood values for the COI gene in contrast to only

minor shifts in tree topology in case of Ef1α. The significant result of the partition homogeneity

test can be explained by the high degree of saturation and therefore the presence of noise in the

COI gene. Dolphin et al. (2000) found that results of the ILD test are likely to be unreliable when

comparing a partition with high degree of noise to one with low noise. The high number of trees

found in parsimony analyses of the Ef1α gene can be attributed to the lack of informative

characters in recent species splits which can therefore not be resolved. In combined analyses the

consistency index and retention index of combined trees are lower than for trees based on Ef1α

sequences, but higher than for trees calculated from the COI gene. While this reflects the pattern

15

one would expect from the higher degree of homoplasy being present in the COI gene as opposed

to Ef1α it has to be noted that the CI and RI are not particularly well suited as a measure of fit for

trees based on DNA sequences as these measures do not reflect the probability of recovering the

correct tree by maximum parsimony (Yang 2006). Combined analysis also reduced the number of

most parsimonious trees to 26 compared to 520421 for Ef1α and 168 for COI single gene

analyses. Support values in combined analyses are either roughly equal or higher than in single

gene analyses of Ef1α except for young nodes where COI provides resolution not present in the

Ef1α gene. We therefore accept the total evidence approach to provide the optimal combination

of resolving power of the two genes. Accordingly our discussion is mostly based on the combined

dataset, and we make reference to Ef1α gene trees only whenever significant differences were

detected. Low support values in combined analyses are predominantly observed on short internal

branches that could in many cases only be resolved by Bayesian analyses. Resolving temporally

closely spaced basal splits (i.e. when the group experienced a high rate of diversification) is a

common issue that has been observed in a large number of phylogenetic studies. This lack of

branch support is likely caused by an insufficient number of informative characters acquired

between temporally closely spaced splitting events. We also recognize that Bayesian posterior

probabilities may overestimate phylogenetic support (Suzuki et al. 2002; Simmons et al. 2004;

but see Wilcox et al. 2002; Alfaro et al. 2003); therefore we limit discussion to relationships that

have adequate bootstrap support in addition to high support from Bayesian posterior probabilities.

The amount of variation found within Ef1α sequences among Eois and the high degree of

mutational saturation of COI sequences suggest that Eois is a broadly defined genus when

compared to other Lepidopteran genera (e.g. Brower et al. 2006; Mallarino et al. 2006;

Hundsdoerfer et al. 2009). The average distance of Ef1α sequences within Eois exceeds the value

in the sample of ithomiine butterflies used by Brower et al. (2006). This fits well with the diverse

morphology of Eois moths. This is not an unexpected result and is probably the case with

numerous, especially tropical, moth genera. The average COI sequence divergence (8.7%) is also

well above the mean of 6.6% found by Hebert et al. (2003) for COI distances between congeneric

species in Lepidoptera and is probably capped by saturation effects.

The monophyly of Eois was always well supported. However, due to our limited sampling of

outgroup taxa we cannot firmly exclude that more extensive sampling of other Neotropical

Larentiinae might reveal Eois not to be truly monophyletic. We consider that to be unlikely for

three reasons: 1. No potential candidates are apparent to us in a sample of ~2000 Neotropical

16

geometrid species (Brehm et al., 2005, 2007); rather some species traditionally included in Eois

(Scoble 1999) turned out to be misplaced in that genus upon closer taxonomic scrutiny (G. Brehm,

unpublished results); 2. It would be a most unlikely coincidence if Old World Eois were much

more closely related, especially when branch lengths are considered next to topology, to

Neotropical Eois than to any of the other included Larentiinae; 3. The monophyly of Eois is not

an unexpected result but rather a confirmation of prior taxonomic hypotheses which subsequently

gained additional support from female genitalia morphology (Holloway, 1997). Old World Eois

are more similar in their wing patterns and general appearance to Neotropical Eois than any other

Neotropical geometrid known to us. The monophyly of Neotropical Eois was not always

unequivocally supported. ML analysis recovered Neotropical Eois as paraphyletic albeit without

sufficient bootstrap support. Additional sampling of both Old and New World Eois could reveal

that either of the two groups is not actually monophyletic.

Phylogenetic signal in wing patterns

Subdivision of Eois into stable monophyletic clades largely coincides with clearly separable wing

pattern types, which we therefore designated as informal species groups. According to our

phylogenetic hypothesis, all such wing pattern types evolved only once with the possible

exception of the chrysocraspedata type (brown with yellow margins). The wing patterns of the

catana clade closely resemble those in the chrysocraspedata clade. A similar pattern type is again

found in E. plumbacea, an Old World species (Holloway 1997). This brown-yellow wing pattern

may therefore either be the plesiomorphic character state for Eois as a whole, or a case of

convergent evolution (photographs are presented in Fig. 2). To finally settle this question more

material (both from the Old World and New World) needs to be analyzed. Notably, many

Neotropical species in the unrelated genus Tricentra (subfamily Sterrhinae) display a strikingly

similar pattern. In Asia, certain species of the genus Chrysocraspeda (Sterrhinae) also exhibit a

pattern virtually undistinguishable from that found in Eois (Plate 1 in Holloway 1997). Although

this seems to be a common pattern its biological significance, if any, is entirely unknown. Wing

shapes within Eois were found to be unusually variable, as pointed and round wings are found in

the same clade and even among closely related species (e.g. within the chrysocraspedata clade).

In conclusion wing pattern morphology is mostly concordant with phylogenetic relationships.

Additionally, a high degree of cryptic diversity was revealed in a number of species complexes,

see Strutzenberger et al. (2010) for a detailed integrative taxonomy based analysis of species

diversity of Neotropical Eois.

17

Host plant associations

When all available food plant records for Eois are mapped on the tree (Figs. 1 and S5,6) it

becomes apparent that affiliations with host plants in the family Piperaceae are widespread within

Eois. A switch of larval host plant from Piper to Peperomia, which is the extant sister group of

Piper (Smith et al., 2008), does probably require only very limited novel physiological adaptation.

It is therefore not surprising that feeding on Peperomia occurs in at least two independent

instances and may well be present in multiple independent lineages of Eois. The same is probably

true for feeding on Manekia. In a molecular phylogenetic study (Wanke et al. 2006) Manekia was

recovered as the first branching clade of Piperaceae. While this indicates that Manekia is more

distantly related to Piper than Peperomia it is unlikely that a high degree of physiological

adaptation was necessary to enable the use of Manekia as larval host plants. However, the

phytochemistry of Manekia is still entirely unknown. The finding that E. chasca also feeds on

Alloplectus sp. (besides Peperomia) is unexpected as the Gesneriaceae do not share

phytochemical similarities with the other known Eois host plant families. At the current state, the

record on Gesneriaceae appears to be singular (Dyer et al. 2009), and it cannot be ruled out that it

represents only a stray larva. Further records on Gesneriaceae are therefore required in order to

confirm such an association. Affiliations with Chloranthaceae are currently confined to the

adimaria clade, which is likely to be the ancestral state for this clade. The Chloranthaceae were

once considered to belong to the Piperales but are currently placed as sister to the entire

magnoliids (Stevens 2001) which contain the Piperales. One Eois species belonging to the

chrysocraspedata clade was found to feed on Siparuna pyricarpa (Monimiaceae), another family

within the magnoliids (Stevens 2001). As in the case of Gesneriaceae, further records will have to

be gathered to determine the significance of the association with Monimiaceae. Feeding on

Hedyosmum, Peperomia and Monimiaceae occurs in taxa that are quite closely related to known

Piper feeders. Both available native host plant records of Asiatic representatives of Eois refer to

Euphorbiaceae (Miller et al. 2009; Singh 1953).

According to our results the ancestral food plant taxa were either Piperaceae or Euphorbiaceae.

Novotny et al. (2003) found one unidentified species of Eois to feed on the invasive Piper

umbellatum in New Guinea. This observation leaves one to speculate that the switch from

Piperaceae to Euphorbiaceae or vice versa did not require a high degree of physiological

adaptation. Notably, Mallotus (Euphorbiaceae), which was found to be the larval host plant of E.

18

grataria (Singh 1953), is similar to the invasive Piper umbellatum in some aspects of its biology

(e.g. both are woody shrubs that grow in disturbed edge habitats and produce new leaves almost

all the year round). The herbivore fauna recorded by Novotny et al. (2003) from invasive Piper

species in New Guinea is mostly comprised of polyphagous insects that otherwise feed on a range

of shrubs and trees in secondary growth, where pioneer euphorbs such as Mallotus and

Macaranga play a prominent role. The most parsimonious explanation for the pattern of host

plant use observed within Neotropical Eois (Fig. S5) is currently that feeding on Piper is the

plesiomorphic state as it is already present in early branching clades of Neotropical Eois.

Independent switches to Peperomia occurred in the odatis and the chasca clade, and one switch

to Monimiaceae in the chrysocraspedata clade. Feeding on Hedyosmum either evolved at least

twice within the adimaria clade, or part of the clade reverted to Piper feeding. Available data do

not allow to firmly decide between these competing hypotheses. Our analyses show that several

switches between larval host plant families have occurred within Eois. While this suggests that

chemical barriers against these host switches were not very high there is nothing yet known on

the physiological effects and demand the secondary compounds of those plant groups pose on

Eois larvae. Chemically very different compounds may well have similar effects and require

similar strategies for detoxification.

Biogeography

The recovered relationships are compatible with the origin of Eois being either in the Old World

or in the Neotropics followed by transoceanic dispersal. Gondwana relationships can be excluded

as the origin of Macrolepidoptera postdates the split of Gondwana by at least 20Myr (Grimaldi &

Engel 2005). To gain further insight into the geographic origin the sister group of Eois needs to

be established with confidence. The single African species of Eois represented in our study is

nested within Asiatic Eois. With only one African species represented in our trees no conclusions

can be drawn on the biogeographic history within the Old World clade. Central and South

American Eois are most likely the result of a single Neotropical colonization as separate

colonizations would imply a high degree of morphological convergence in numerous cases. This

is also supported in our analyses where the two Costa Rican species are not sister to each other

but are associated with morphologically similar species from Ecuador. Sampling of more Central

American representatives will be required to further corroborate, or refute, this hypothesis.

Outgroup relationships

19

We recovered two, moderately supported, alternative relationships between Eois and the

remaining Larentiinae. Firstly, MP and ML trees show Eois as the sister group to all other

included Larentiinae except for D. abortivaria, among them two members of Eupithecia and one

further species of Eupitheciini (Pasiphila rectangulata). We were unable to confirm the tribal

placement of Holloway (1997) as Eois was never recovered in a clade together with any

Eupitheciini. Therefore, our analyses give no support for a close relationship between this latter

enormously diverse geometrid tribe and the genus Eois. Secondly, the association of Eois with

Philereme transversata as recovered in Bayesian analyses and ML trees of the Ef1α gene is

unexpected when considering the morphology and ecology of Philereme in contrast to Eois. In

light of the poor phylogenetic resolution within the Larentiinae and the limited taxon sampling

that was available to us these results should be interpreted with great care. For future analyses a

more thorough sampling of Larentiinae tribes, including Neotropical and Palaeotropical taxa, is

desirable. In addition to that, the instability of relationships and often poor support values within

the outgroup indicate that a larger sequence dataset will be necessary to obtain a robust placement

of Eois within the subfamily. The position of Dyspteris abortivaria was unexpected and does not

unequivocally confirm the assignment of Dyspteris to the Larentiinae (Scoble 1999).

Conclusion

The approach taken in this study has proven to be very successful in reconstructing a well-

resolved initial phylogenetic hypothesis of Eois. For the first time could the large morphological

diversity within this genus be placed into a solid phylogenetic framework which will be of vital

help in ongoing research on the ecology and biodiversity of Neotropical Eois. It is also a

substantial step towards forthcoming taxonomic revisions of Neotropical Eois. Our study has

shown that Eois is not only an interesting target group for ecological research but also a

promising target for closer investigation of its evolutionary history. Future challenges will be the

extension of geographic sampling, gathering further host plant data and the generation of

additional datasets (i.e. larval parasitoids, morphometric data, gentitalic anatomy) that can only be

validly analyzed in a phylogenetic context.

Acknowledgements

20

We thank Manuela Zimmermann for providing the majority of moths used in this work; Christian

H. Schulze and Terence M. Whitaker for providing Eois from Borneo; Hermann Staude for

providing Eois from Africa; Christine Truxa for supplying moths from Austria for use as

outgroup taxa; Brigitte Gottsberger for assistance in the molecular lab; Martin Wiemers for

helpful discussions on data analysis; Aleksandros Stamatakis for advice on the use of RAxML.

Jürgen Homeier and Eric Tepe provided identifications of Eois host plants recorded in southern

Ecuador. This study was financially supported by grants from the Deutsche

Forschungsgemeinschaft (FOR 402, Fi 547/6-3; FOR 816, Fi 547/10-1). The foundation Nature

and Culture International (Loja/Ecuador, Del Mar/USA) allowed access to their property for field

work. The Ministerio del Ambiente (Ecuador) kindly issued the necessary research permit (002-

PNP-DBAP-RLZCH/MA), and the authorities in Austria provided collecting permits for the

national parks “Donauauen” and “Gesäuse”, respectively.

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26

Figure legends:

Figure 1. Bayesian 50% consensus tree of Eois based on total evidence with Bayesian posterior

probabilities (above), ML bootstrap values (bottom left) and MP bootstrap values (bottom right)

added to branches. Host plant records are indicated on species and clade level. An image of a

representative member is given for each clade in order of appearance of the clades in the tree.

Geographic origin of taxa is given for taxa not collected in Ecuador. Ancestral states of food plant

use for selected nodes are indicated whenever MacClade provided an unequivocal reconstruction.

For three nodes, where maximum likelihood reconstruction of ancestral states provided additional

information, pie charts representing the likelihood of each state are displayed.

Figure 2. Bayesian 50% consensus tree of the outgroups with Bayesian posterior probabilities

(above), ML bootstrap values (bottom left) and MP bootstrap values (bottom right) added to

branches.

Figure S1. Plots of Ti/Tv ratio against pairwise distances for Ef1α (top) and COI (bottom) for 1st

and 2nd codon positions, and separately for 3rd codon positions.

Figure S2. Bayesian 50% consensus tree of the Ef1α gene, values on branches are Bayesian

posterior probabilities. Asterisk indicates Bayesian posterior probabilities of 1.00.

Figure S3 Bayesian 50% consensus tree of the COI gene, values on branches are Bayesian

posterior probabilities. Asterisk indicates Bayesian posterior probabilities of 1.00.

Figure S4. a: Best ML tree recovered from total evidence analyses with bootstrap values indicated.

b: Strict consensus of 26 MP trees recovered from total evidence analyses with bootstrap values

indicated.

Figure S5. Maximum parsimony optimized mapping of host plant characters on the Bayesian tree.

Numbers next to the figure legend indicate the applied coding scheme for host plant associations.

Squares between branches and taxon names indicate taxa for which a host plant association was

coded in the character matrix; the states of all other taxa are inferred. For a detailed list of host

plant records and how they were coded see Table S1.

27

Figure S6. Maximum likelihood reconstruction of host plant character states using the total

evidence Bayesian tree topology. Numbers next to figure legend indicate the applied coding

scheme for host plant associations. Pie charts at nodes indicate relative probabilities of each state

for the respective node.

E. spnr borrata 396 Eo00181 E. spnr borrata 396 Eo00282 E. spnr borrata 396 Eo00333

E. borrata 396 Eo00518 E. sp. 400 Eo00350

E. spnr adimaria 399 Eo00188 E. spnr adimaria 399 Eo00537

E. spnr margarita Eo00002 E. spnr margarita Eo00010

E. spnr golosata Eo00219 E. spnr golosata Eo00012

E. spnr golosata Eo00028 E. spnr golosata Eo00307

E. sp. Eo00095 E. sp. 385 Eo00080

E. sp. 977 Eo00187 E. escamata 390 Eo00035

E. spnr azafranata Eo00154 E. spnr azafranata Eo00281

E. sp. Eo00533 E. sp. Eo00353

E. sp. Eo00489 E. spnr. lucivittata 384 Eo00283

E. sp. 420 Eo00205 E. spnr telegraphica Eo00041

E. sp. Eo00082 E. spnr chasca Eo00107

E. chasca 392 Eo00096 E. spnr inflammata 402 Eo00060

E. antiopata 799 Eo00023 E. spnr inflammata 515 Eo00098

E. sp. Eo00339 E. planetaria 383 Eo00150

E. spnr encina Eo00149 E. spnr pallidicosta Eo00217

E. spnr pallidicosta Eo00239 E. spnr pallidicosta 1023 Eo00244

E. sp. 409 Eo00063 E. spnr ignefumata 389 Eo00143

E. spnr ignefumata 30 Eo00510 E. spnr paraviolascens Eo00065 E. spnr paraviolascens 423 Eo00190

E. sp. Eo00066 E. sp. 425 Eo00040

E. spnr paraviolascens Eo00024 E. spnr chrysocraspedata 1029 Eo00077

E. spnr chrysocraspedata Eo00476 E. spnr muscosa 803 Eo00045

E. sp. 1070 Eo00103 E. spnr goodmanii Eo00497

E. spnr goodmanii Eo00526 E. spnr goodmanii Eo00144 E. spnr goodmanii Eo00014 E. spnr goodmanii Eo00044 E. spnr goodmanii Eo00133

E. spnr goodmanii Eo00231 E. spnr olivacea Eo00031 E. spnr olivacea Eo00085 E. spnr olivacea Eo00087

E. spnr olivacea Eo00263 E. spnr olivacea Eo00480

E. spnr olivacea 408 Eo00090 E. spnr olivacea Eo00335

E. sp. 1041 Eo00477 E. multistrigaria 1686 Eo00386 COSTA RICA

E. sp. Eo00481 E. sp 820 Eo00048

E. sp. 836 Eo00016 E. sp. Eo00349

E. spnr odatis 419 Eo00073 E. spnr nigrosticta 388 Eo00340

E. angulata 376 Eo00255 E. spnr nigrinotata Eo00180

E. spnr nigrinotata Eo00218 E. sp. 386 Eo00003

E. sp. 394 Eo00163 E. spnr nigrinotata Eo00245

E. sp. 2128 Eo00323 E. sp. 961 Eo00099 E. sp. 961 Eo00139

E. biradiata 410 Eo00008 E. spnr biradiata Eo00227

E. spnr lunifera 395 Eo00125 E. sp. 405 Eo00173

E. spnr violada Eo00034 E. spnr violada 403 Eo00097

E. spnr violada Eo00381 E. spnr lunifera Eo00331

E. spnr guapa Eo00520 E. sp. 2129 Eo00326

E. sp. Eo00290 E. sp. Eo00511

E. spnr camptographata 739 Eo00345 E. isographata 1433 Eo00382 COSTA RICA

E. spnr sagittaria 377 Eo00166 E. spnr concatenata Eo00214

E. spnr concatenata Eo00238 E. sp. 427 Eo00019

E. sp. 411 Eo00042 E. spnr trillista 441 Eo00111

E. sp. Eo00226 E. sp. Eo00499

E. spnr. restrictata 837 Eo00504 E. spnr serrilineata Eo00013

E. spnr catana 426 Eo00352 E. serrilineata Eo00488

E. plumbacea Eo00414 BORNEO E. sp. Eo00463 SOUTH AFRICA

E. pheneroscia Eo00424 BORNEO E. menorata Eo00410 BORNEO E. pallidula Eo00417 BORNEO E. obliviosa Eo00466 BORNEO

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.001.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.001.00

1.00

1.00

1.00

1.00

1.00

1.00

0.99

1.001.00

1.00

1.00

0.98

1.00

0.2

100/100

1.00100/94

1.0093/92

0.59--/--

0.61--/--

0.78--/--

99/65

100/100

1.0091/96

100/100

1.00100/100

0.9999/96

1.0096/100

1.0089/--

1.0098/50

0.80--/--

98/95

0.5267/75

0.9884/83

1.0099/90

100/83

0.9786/--

100/97

0.9863/55

0.8950/--

1.0094/71 99/89

0.54--/--

0.93--/--

0.64--/--

99/97

0.90--/--

1.0091/69

100/1001.00

93/99 100/99

0.9563/--

100/100

1.0095/64

1.0086/57

100/100

0.8359/--

100/98

100/99

1.0091/67

0.64--/--

100/87 100/100

1.0069/--

1.0092/76

0.8957/--

0.8551/--

100/89

0.89--/--

100/98

100/100

1.0084/87

1.0087/52

0.9069/53

1.0068/--

0.59--/--

100/97

100/100

1.0085/92

0.58--/--

0.90--/-- 100/100

100/100

1.0090/55

1.00100/1001.00

100/100

0.8872/69

1.0085/62

1.0097/93

1.0081/69

0.5657/--

100/100

100/1001.00

99/88

100/991.0096/62

1.0088/53

0.79--/--

0.9970/68

1.0077/74

0.9667/--

0.73--/--

0.59--/--

95/51

90/83

100/83100/100

1.0073/--

100/990.75

58/70 94/79

100/97

100/1000.83

53/59

--/64

100/98

93/65

99/99

99/95100/96

95/99

1.00100/100

adimaria clade

chasca clade

pallidicosta clade

chrysocraspedata clade

olivacea clade

odatis clade

sagittaria clade

trillista clade

Old World clade

catana clade

P

H

Pe

Piper (Piperaceae)

Hedyosmum (Chloranthaceae)

Peperomia (Piperaceae)

Outgroup

E Euphorbiaceae

E. spnr catana

E. pallidicosta

E. spnr sagittaria

G

M

Peperomia + Gesneriaceae

Monimiaceae

E. plumbacea

S Manekia (=Sarcorhachis) (Piperaceae)

E. spnr adimaria

E. chasca

E. spnr chrysocraspedata

E. olivacea

E. spnr odatis

E. spnr trillista

HHH

H

HH

HH

HH

PP

P

PP

PP

P

PP

P

PP

PP

PP

PP

PPP

PPP

PP

P

P

PP

PP

P

PP

P

P

P

P

G

Pe

Pe

S

P

P

P

P

P

P

P

M

H

G

M

Pe

E P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

E

P

P

P

Figure 1

Philereme transversata Eo00393 Pasiphila rectangulata Eo00401

Eupithecia linariata Eo00402 Eupithecia impurata Eo00405

Ecliptopera silaceata Eo00391 Eulithis pyraliata Eo00398

Perizoma minoratum Eo00407 Hydriomena impluviata

Xanthorhoe quadrifasciata Eo00395 Phibalapteryx virgata

Epirrhoe alternata Eo00396 Epirrhoe hastulata

Anticollix sparsata Trichopteryx carpinata

Dyspteris abortivaria Rhodostrophia calabra

Rhodostrophia vibicaria Scopula decorata

Idaea aversata Idaea straminata

Cyclophora albipunctata Cyclophora punctaria

Timandra comae Timandra griseata

Haematopis grataria Rhodometra sacraria

Lythria cruentaria Lythria purpuraria

Lythria sanguinaria Archiearis parthenias

1.00

1.001.00

1.001.00

0.94

1.00

1.00

1.001.00

0.78

1.00

0.89

1.001.00

1.00

0.96

0.96

1.00

0.98

1.00

1.00

0.99

0.2

--/--

0.96--/--

--/--

--/--

92/68

78/--

98/67100/100

100/100

73/92

94/74

73/54

75/--100/100

0.7254/--

100/100

100/100100/100

100/100

100/99

0.9454/--

0.99--/--

1.00100/--

85/--59/--

--/--

--/--

74/--

Eois

Larentiinae

Sterrhinae

Figure 2

Table 1. List of all taxa used in phylogenetic analyses (in alphabetical order within subfamilies) with collection

sites (for own sequences only) and Genbank accession numbers indicated. Numeric species codes of Eois

specimens are given in the Taxon column behind species names for reference to G. Brehm's species database

(Brehm et al., 2005, electronic supplement) when taxa could be matched with confidence. Code: reference

number assigned to each specimen before sequencing. Collection site: specimens from Ecuador come from

various sites in and around the Reserva Biológica San Francisco (RBSF) region and surroundings (see Material

and Methods section). Site codes with Q refer to ravine forest at 1900-2100 m asl (Günter et al., 2008).

Bombuscaro: lower montane forest at 1020 m asl (Brehm et al., 2003b). Cajanuma: upper montane forest.

C=Canfield et al., 2008; K=Knoelke et al., 2004; O=Ounap et al., 2008; S=Snäll et al., 2007; T=this study;

W=Wahlberg et al., 2005.

Taxon Code Collection site COI Ref. Ef1a Ref. Archiearinae Archiearis parthenias DQ018928 W DQ018899 W Larentiinae Anticollix sparsata EU443353 O EU443291 O Dyspteris abortivaria EU151592 C EU151695 C Ecliptopera silaceata Eo00391 Orth a. d. Donau, Austria GQ433611 T GQ433490 T Eois angulata 376 Eo00255 Q5#1 RBSF, Ecuador GQ433567 T GQ433446 T Eois antiopata 799 Eo00023 Q2#3 RBSF, Ecuador GQ433508 T GQ433387 T Eois biradiata 410 Eo00008 Q3#3 RBSF, Ecuador GQ433501 T GQ433380 T Eois borrata 396 Eo00518 Bombuscaro, Ecuador GQ433600 T GQ433479 T Eois chasca 392 Eo00096 Q5#3 RBSF, Ecuador GQ433532a T GQ433411 T Eois escamata 390 Eo00035 Q2#2 RBSF, Ecuador GQ433513 T GQ433392 T Eois menorata Eo00410 Sabah, Kinabalu NP, Borneo GQ433605 T GQ433484 T Eois multistrigaria 1686 Eo00386 Braulio Carillo NP, Costa Rica GQ433588 T GQ433467 T Eois obliviosa Eo00466 Sabah, Imbak Canyon, Borneo GQ433610 T GQ433489 T Eois pallidula Eo00417 Sabah, Kinabalu NP, Borneo GQ433607 T GQ433486 T Eois pheneroscia Eo00424 Sabah, Kinabalu NP, Borneo GQ433608 T GQ433487 T Eois planetaria 383 Eo00150 Q3#2 RBSF, Ecuador GQ433545 T GQ433424 T Eois plumbacea Eo00414 Sabah, Kinabalu NP, Borneo GQ433606 T GQ433485 T Eois serrilineata Eo00488 Bombuscaro, Ecuador GQ433593 T GQ433472 T Eois sp. Eo00066 Q2#2 RBSF, Ecuador GQ433523 T GQ433402b T Eois sp. Eo00082 Q3#2 RBSF, Ecuador GQ433527 T GQ433406 T Eois sp. Eo00095 Q5#3 RBSF, Ecuador GQ433531 T GQ433410 T Eois sp. Eo00226 Q3#1 RBSF, Ecuador GQ433560 T GQ433439 T Eois sp. Eo00290 Q3#3 RBSF, Ecuador GQ433572 T GQ433451 T Eois sp. Eo00339 BC-IV RBSF, Ecuador GQ433579 T GQ433458 T Eois sp. Eo00349 BC-VI RBSF, Ecuador GQ433582 T GQ433461 T Eois sp. Eo00353 4a RBSF, Ecuador GQ433585 T GQ433464a T

Eois sp. Eo00463 Oribi Gorge Nature Reserve, South Africa GQ433609 T GQ433488 T

Eois sp. Eo00481 Bombuscaro, Ecuador GQ433592 T GQ433471 T Eois sp. Eo00489 Bombuscaro, Ecuador GQ433594 T GQ433473 T Eois sp. Eo00499 Bombuscaro, Ecuador GQ433596 T GQ433475 T Eois sp. Eo00533 RBSF, 2670m, Ecuador GQ433603 T GQ433482 T Eois sp. Eo00511 Bombuscaro, Ecuador GQ433599 T GQ433478 T Eois sp. 1041 Eo00477 Bombuscaro, Ecuador GQ433590 T GQ433469 T Eois sp. 1070 Eo00103 Q5#2 RBSF, Ecuador GQ433536 T GQ433415 T Eois sp. 2128 Eo00323 Q3#2 RBSF, Ecuador GQ433574 T GQ433453 T Eois sp. 2129 Eo00326 Q2#3 RBSF, Ecuador GQ433575 T GQ433454 T Eois sp. 385 Eo00080 Q2#2 RBSF, Ecuador GQ433526 T GQ433405 T Eois sp. 386 Eo00003 Q2#2 RBSF, Ecuador GQ433500 T GQ433379 T Eois sp. 394 Eo00163 Q3#1 RBSF, Ecuador GQ433547 T GQ433426 T Eois sp. 400 Eo00350 BC-VI RBSF, Ecuador GQ433583 T GQ433462 T

Eois sp. 405 Eo00173 Q3#3 RBSF, Ecuador GQ433549 T GQ433428 T Eois sp. 409 Eo00063 Q2#2 RBSF, Ecuador GQ433521 T GQ433400 T Eois sp. 411 Eo00042 Q2#3 RBSF, Ecuador GQ433516 T GQ433395 T Eois sp. 420 Eo00205 Q3#1 RBSF, Ecuador GQ433555 T GQ433434 T Eois sp. 425 Eo00040 Q3#1 RBSF, Ecuador GQ433514 T GQ433393 T Eois sp. 427 Eo00019 Q3#1 RBSF, Ecuador GQ433507 T GQ433386 T Eois sp. 820 Eo00048 Q3#3 RBSF, Ecuador GQ433519 T GQ433398 T Eois sp. 836 Eo00016 Q3#1 RBSF, Ecuador GQ433506 T GQ433385 T Eois sp. 961 Eo00099 Q2#3 RBSF, Ecuador GQ433535 T GQ433414 T Eois sp. 961 Eo00139 Q2#3 RBSF, Ecuador GQ433541 T GQ433420 T Eois sp. 977 Eo00187 Q5#3 RBSF, Ecuador GQ433552 T GQ433431 T Eois spnr adimaria 399 Eo00188 Q5#3 RBSF, Ecuador GQ433553 T GQ433432 T Eois spnr adimaria 399 Eo00537 RBSF, 2670m, Ecuador GQ433604 T GQ433483 T Eois spnr azafranata Eo00154 Q2#3 RBSF, Ecuador GQ433546 T GQ433425 T Eois spnr azafranata Eo00281 Q3#3 RBSF, Ecuador GQ433569 T GQ433448 T Eois spnr biradiata Eo00227 Q3#1 RBSF, Ecuador GQ433561 T GQ433440 T Eois spnr borrata 396 Eo00181 Q2#3 RBSF, Ecuador GQ433551 T GQ433430 T Eois spnr borrata 396 Eo00282 Q3#3 RBSF, Ecuador GQ433570 T GQ433449 T Eois spnr borrata 396 Eo00333 Q5#2 RBSF, Ecuador GQ433577 T GQ433456 T Eois spnr camptographata 739 Eo00345 BC-II RBSF, Ecuador GQ433581 T GQ433460 T Eois spnr catana 426 Eo00352 4a RBSF, Ecuador GQ433584 T GQ433463 T Eois spnr chasca Eo00107 Q5#3 RBSF, Ecuador GQ433537 T GQ433416a T Eois spnr chrysocraspedata Eo00476 Bombuscaro, Ecuador GQ433589 T GQ433468 T Eois spnr chrysocraspedata 1029 Eo00077 Q2#2 RBSF, Ecuador GQ433525 T GQ433404 T Eois spnr concatenata Eo00214 Q3#3 RBSF, Ecuador GQ433556 T GQ433435 T Eois spnr concatenata Eo00238 Q5#2 RBSF, Ecuador GQ433563 T GQ433442 T Eois spnr encina Eo00149 Q3#2 RBSF, Ecuador GQ433544 T GQ433423 T Eois spnr golosata Eo00012 Q3#3 RBSF, Ecuador GQ433503 T GQ433382 T Eois spnr golosata Eo00028 Q3#2 RBSF, Ecuador GQ433510 T GQ433389a T Eois spnr golosata Eo00219 Q3#2 RBSF, Ecuador GQ433559 T GQ433438a T Eois spnr golosata Eo00307 Q5#2 RBSF, Ecuador GQ433573 T GQ433452 T Eois spnr goodmanii Eo00014 Q3#3 RBSF, Ecuador GQ433505 T GQ433384 T Eois spnr goodmanii Eo00044 Q2#3 RBSF, Ecuador GQ433517 T GQ433396 T Eois spnr goodmanii Eo00133 Q2#3 RBSF, Ecuador GQ433540 T GQ433419 T Eois spnr goodmanii Eo00144 Q3#2 RBSF, Ecuador GQ433543 T GQ433422 T Eois spnr goodmanii Eo00231 Q5#1 RBSF, Ecuador GQ433562 T GQ433441 T Eois spnr goodmanii Eo00497 Bombuscaro, Ecuador GQ433595 T GQ433474 T Eois spnr goodmanii Eo00526 Bombuscaro, Ecuador GQ433602 T GQ433481 T Eois spnr guapa Eo00520 Bombuscaro, Ecuador GQ433601 T GQ433480 T Eois spnr ignefumata 30 Eo00510 Bombuscaro, Ecuador GQ433598 T GQ433477 T Eois spnr ignefumata 389 Eo00143 Q5#2 RBSF, Ecuador GQ433542 T GQ433421 T Eois spnr inflammata 402 Eo00060 Q3#3 RBSF, Ecuador GQ433520 T GQ433399 T Eois spnr inflammata 515 Eo00098 Q2#3 RBSF, Ecuador GQ433534 T GQ433413b T Eois spnr isographata 1433 Eo00382 Braulio Carillo NP, Costa Rica GQ433587 T GQ433466 T Eois spnr lilacea telegraphica Eo00041 Q2#3 RBSF, Ecuador GQ433515 T GQ433394 T Eois spnr lucivittata 384 Eo00283 Q3#3 RBSF, Ecuador GQ433571 T GQ433450a T Eois spnr lunifera Eo00331 Q5#2 RBSF, Ecuador GQ433576 T GQ433455 T Eois spnr lunifera 395 Eo00125 Q3#3 RBSF, Ecuador GQ433539 T GQ433418 T Eois spnr margarita Eo00002 Q3#3 RBSF, Ecuador GQ433499 T GQ433378 T Eois spnr margarita Eo00010 Q3#3 RBSF, Ecuador GQ433502 T GQ433381 T Eois spnr muscosa 803 Eo00045 Q2#3 RBSF, Ecuador GQ433518 T GQ433397 T Eois spnr nigrinotata Eo00180 Q2#3 RBSF, Ecuador GQ433550 T GQ433429 T Eois spnr nigrinotata Eo00218 Q5#3 RBSF, Ecuador GQ433558 T GQ433437 T Eois spnr nigrinotata Eo00245 Q5#3 RBSF, Ecuador GQ433566 T GQ433445 T Eois spnr nigrosticta 388 Eo00340 BC-I RBSF, Ecuador GQ433580 T GQ433459 T Eois spnr odatis 419 Eo00073 Q3#3 RBSF, Ecuador GQ433524 T GQ433403 T Eois spnr olivacea Eo00031 Q2#1 RBSF, Ecuador GQ433511 T GQ433390 T Eois spnr olivacea Eo00085 Q2#3 RBSF, Ecuador GQ433528 T GQ433407 T Eois spnr olivacea Eo00087 Q2#3 RBSF, Ecuador GQ433529 T GQ433408 T Eois spnr olivacea Eo00263 Q2#3 RBSF, Ecuador GQ433568 T GQ433447 T Eois spnr olivacea Eo00335 BC-IV RBSF, Ecuador GQ433578 T GQ433457 T Eois spnr olivacea Eo00480 Bombuscaro, Ecuador GQ433591 T GQ433470 T Eois spnr olivacea 408 Eo00090 Q2#1 RBSF, Ecuador GQ433530 T GQ433409 T Eois spnr pallidicosta Eo00217 Q5#3 RBSF, Ecuador GQ433557 T GQ433436a T Eois spnr pallidicosta Eo00239 Q5#2 RBSF, Ecuador GQ433564 T GQ433443a T Eois spnr pallidicosta 1023 Eo00244 Q5#3 RBSF, Ecuador GQ433565 T GQ433444b T Eois spnr paraviolascens Eo00024 Q2#3 RBSF, Ecuador GQ433509 T GQ433388 T Eois spnr paraviolascens Eo00065 Q2#2 RBSF, Ecuador GQ433522 T GQ433401 T Eois spnr paraviolascens 423 Eo00190 Q2#1 RBSF, Ecuador GQ433554 T GQ433433 T Eois spnr restrictata 837 Eo00504 Bombuscaro, Ecuador GQ433597 T GQ433476 T

Eois spnr sagittaria 377 Eo00166 Q2#3 RBSF, Ecuador GQ433548 T GQ433427 T Eois spnr serrilineata Eo00013 Q3#3 RBSF, Ecuador GQ433504 T GQ433383 T Eois spnr trillista 414 Eo00111 Q2#3 RBSF, Ecuador GQ433538 T GQ433417 T Eois spnr violada Eo00034 Q2#2 RBSF, Ecuador GQ433512 T GQ433391 T Eois spnr violada Eo00381 4a RBSF, Ecuador GQ433586 T GQ433465 T Eois spnr violada 403 Eo00097 Q2#3 RBSF, Ecuador GQ433533 T GQ433412 T Epirrhoe alternata Eo00396 Orth a. d. Donau, Austria GQ433614 T GQ433493 T Epirrhoe hastulata EU443351 O EU443289 O Eulithis pyraliata Eo00398 Orth a. d. Donau, Austria GQ433615 T GQ433494 T Eupithecia impurata Eo00405 Gesäuse NP, Austria GQ433618 T GQ433497 T Eupithecia linariata Eo00402 Gesäuse NP, Austria GQ433617 T GQ433496 T Hydriomena impluviata EU443350 O EU443288 O Lythria cruentaria EU443365 O EU443302 O Lythria purpuraria EU443367 O EU443304 O Lythria sanguinaria EU443366 O EU443303 O Pasiphila rectangulata Eo00401 Orth a. d. Donau, Austria GQ433616 T GQ433495 T Perizoma minoratum Eo00407 Gesäuse NP, Austria GQ433619 T GQ433498 T Phibalapteryx virgata EU443352 O EU443290 O Philereme transversata Eo00393 Orth a. d. Donau, Austria GQ433612 T GQ433491 T Trichopteryx carpinata EU443349 O EU443287 O Xanthorhoe quadrifasciata Eo00395 Orth a. d. Donau, Austria GQ433613 T GQ433492a T Sterrhinae Cyclophora albipunctata EU443360 O EU443297 O Cyclophora punctaria EU443361 O EU443298 O Haematopis grataria EU443364 O EU443301 O Idaea aversata EU443357 O EU443294 O Idaea straminata EU443356 O AY948507 S Rhodometra sacraria AJ870398 K EU443305 O Rhodostrophia calabra EU443355 O EU443293 O Rhodostrophia vibicaria EU443354 O EU443292 O Scopula decorata EU443359 O EU443296 O Timandra comae EU443363 O EU443300 O Timandra griseata EU443362 O EU443299 O

aSequence is shorter than 90% of desired length bEf1α sequence obtained from different individual than COI sequence

Table 2. Summary of character statistics for all partitions used in this study. No. Chars.: Total number of characters; Variable: Number of variable

characters; Informative: Number of parsimony informative characters; Model: Best fitting model of evolution selected by MrModeltest 2.2.

COI COI COI COI COI Ef1α Ef1α Ef1α Ef1α Ef1α all 1st+2nd 1st 2nd 3rd all 1st+2nd 1st 2nd 3rd No. Chars 1536 1024 512 512 512 1225 816 408 408 409

623 152 118 34 471 386 57 36 21 329 Variable 40.56% 14.84% 23.05% 6.64% 91.99% 31.51% 6.99% 8.82% 5.15% 80.44%

543 116 94 22 427 354 45 29 16 309 Informative

35.35% 11.33% 18.36% 4.30% 83.40% 28.90% 5.51% 7.11% 3.92% 75.55% Model GTR+I+G GTR+I+G GTR+I+G GTR+I GTR+G GTR+I+G GTR+I+G GTR+I+G F81+I+G GTR+I+G

Table 3. AIC values and Bayes factors for each partitioning strategy as outlined in table 3. K: Number of

parameters, w: Akaike weight.

-lnL ML K AIC ΔAIC w -lnL

Bayesian Bayes factor

6P 48016.43 335 96702.86 0 1 47782.69 0 4P 48496.68 317 97627.37 924.51 0 47958.26 351.14 3P 49387.88 308 99391.76 2688.90 0 48436.82 1308.26 2P 50449.94 299 101497.89 4795.03 0 49398.39 3231.40 1P 51069.28 290 102718.56 6015.70 0 50433.08 5300.78

Molecular phylogeny of Eois (Lepidoptera, Geometridae): evolution of wing patterns and host plant...

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