1. Introduction

Phytophagous insects and plants make up over half of macroscopic organisms, and the relations between phytophagous insects and their hostplants have been widely discussed. 70-80% of phytophagous insects are considered to be monophagous (feeding on plants within a single genus) or oligophagous (feeding on several plants belonging to different genera within one plant family). This phenomenon provides model relationships for studying herbivore-plant interactions (Chapman, 1982, Bernays and Chapman, 1994). Some of the best examples are butterflies, which at the generic level frequently feed on related hostplants (Ehrlich and Raven, 1964; Jermy, 1984; Fiedler, 1996; Boggs et al., 2003;

Wahlberg et al., 2009). Major evolutionary associations between phytophagous insects and their hostplants have generally been conceived as scenarios of secondary host shifting, replacing the earlier paradigm of cospeciation based on co-phylogenetic approaches (Janz and Nylin, 1998;

Janz et al., 2006). When related insects shift to use another hostplant, it is often based on shared plant secondary chemistry (Carter and Feeny, 1999;

Wahlberg, 2001). For example, many Appias pierids, seek hostplants with mustard oil glucosinolates (Wheat et al., 2007). This has allowed their ancestors to colonize both Malpighiales and Brassicales, orders distantly related to each other (Braby and Trueman, 2006).

Reconstructing phylogenetic relationships of a selected group is an

important step to investigate the mechanism of host shifting to a novel hostplant. Such investigations may sometimes even cast light on plant taxonomy (e.g. the close relationship of the Euphorbiaceae genera

Omphalea and Endospermum: Lees and Smith, 1991; Wurdack and Davis,

2009).

Most butterfly species have diversified on angiosperms (Janz and Nylin, 1998) on which they appear to have started their adaptive radiation back into the late Cretaceous (Wahlberg et al., 2009), and only a small number of butterflies are known to feed on gymnosperms (Ehrlich and Raven, 1964; Beccaloni et al., 2008). These butterflies listed in Table 9 include: Neophasia species (Pieridae) which feed on Pinaceae in North America to Mexico, the hesperiid Metardaris cotsinga on Pinus radiata in Bolivia and some amathusiine nymphalids, such as Faunis aerope,

Taenaris onolaus, and T. butleri, feeding on Cycadaceae in Asia, and one

species of normally monocot-feeding Opsiphanes which feeds on Cycas

circinalis in Venezuela. Some Callophrys butterflies (Lycaenidae) utilize

Pinaceae or Cupressaceae in North America. Other lycaenids utilize the family Cycadaceae such as Theclinesthes onycha (Lycaenidae) in Australia, Eumaeus species in Central America, and some members of

Chilades in Asia.

Considering the phylogenetic position of gymnosperm-feeding butterflies recognized so far (Janz and Nylin, 1998; Braby et al., 2006;

Wahlberg et al., 2009), most of them appear to represent single host colonizations from their distant angiosperm-feeding relatives.

Gymnosperm feeding occurs as recent events in butterflies, whereas it is probably an ancient association in certain groups such as

Araucaria-feeding beetles (Farrell et al., 1998; Sequeira and Farrell, 2001)

and Agathiphagidae moths (Dumbleton, 1952). In two cases mentioned above (Neophasia and Eumaeus), gymnosperm-feeding characterizes the genus, suggesting a relatively old association (as in the case study of

Callioratis and several apparently related genera of cycad-feeding

geometrids: Staude, 2001; Bayliss et al., 2009). In five cases however (Faunis, Taenaris, Callophrys, Theclinesthes and Chilades) the genus also feeds on angiosperms, suggesting a more recent host shift. Species of

Neophasia are only known to feed on gymnosperms so host shifts are

hard to trace without comprehensive phylogenetic analysis of the groups to which they belong, whilst Eumaeus childrenae is also reported from the toxic monocot family Amaryllidaceae and perhaps more doubtfully, E.

atala on Manihot (Ehrlich and Raven, 1964; Contreras-Medina et al.,

2003). However, the fact that different species of Chilades feed on both gymnosperms and angiosperms makes the genus an interesting candidate for examining the pattern of hostshifts and asking how such a dramatic shift was adaptively possible.

In this study, I examine not only the monophyly of Chilades butterflies but also their host associations mapped onto the phylogenetic framework in order to determine origins of Cycas-feeding and frequency of host shifting in the genus Chilades. So far, no morphological or molecular phylogenies are available for Chilades. Only hypothetical genus-relationships have been proposed (Bálint & Johnson, 1997).

Therefore, the main aim of this study is to test the relationships among

Chilades butterflies and their relatives based on the genes mitochondrial

cytochrome oxidase I and II (COI+COII) and nuclear elongation factor 1

alpha (Ef-1α). Information on insect-hostplant associations and geographic distribution were also used to answer how many shifting events happen for Chilades butterflies to use Cycas-plants.

II-2. Materials and methods Taxon sampling

I sampled 82 specimens, representing 13 of 20 Chilades species and 32 related species (Table 1 and 2). Chilades butterflies were sampled representatives from three species groups. One member, C. yunnanensis, not classified to above species groups was also sampled in this study.

Related genera in Polyommatus section sampled here included

Polyommatus, Albulina, Agriades, Plebejus, and Aricia. In addition, the

neotropical genera Echinargus and Hemiargus that were considered to be related to the Polyommatus genus-group (Bálint and Johnson, 1997) were also selected. Moreover, several sections within Polyommatini and

Niphanda fusca (Niphandini) were sampled to find out the most related

genera to Polyommatus genus-group and to test the monophyly of

Chilades.

Molecular methods

The procedures of DNA extraction, PCR, and DNA sequencing reaction were described in Part I. The primers used for amplifying the mitochondrial COI+COII, and nuclear Ef1α genes have been described (Caterino and Sperling, 1999; Kandul et al., 2004), and the novel primers were listed in Table 10.

Sequence treatments

Molecular sequences of COI+COII and Ef1α genes were checked and assembled into contiguous arrays using Sequencher 4.8 (GeneCode, Boston, USA). Coding regions were determined against to the published sequences of Drosophila yakuba (Clary & Wolstenholme, 1985) and

Bombyx mori (Yukuhiro et al., 2002). Primer regions and tRNA-Leu gene

were cropped, and the undetermined first codon of COI and the complete or uncompleted stop codons of COI and COII found in Lepidoptera (Wu et al., 2010a) were also removed in the analyses to avoid length variation.

The data sets were aligned according to amino sequence similarity by ClustalW in MEGA 3.1 (Kumar et al., 2004) with the default settings (gap opening penalty=10, gap extension penalty=0.2). Missing data and ambiguities were designated by “N”.

Phylogenetic analyses

A range of phylogenetic methods was used to infer the phylogeny.

Maximum parsimony (MP) were performed in PAUP*10b (Swofford, 2000). Bayesian inference (BI) was carried out using MrBayes v. 3.1 (Huelsenbeck and Ronquist, 2001; Ronquist and Huelsenbeck, 2003), and Maximum Likelihood (ML) was performed in PHYML (Guindon &

Gascuel, 2003).

To evaluate appropriate outgroup and ingroup combinations for

Chilades butterflies, in the first runs, I used two specimens of Niphanda

fusca as outgroup and the 80 remaining taxa (44 species) as the ingroup

for reconstructing relationships based on MP and BI methods (I refer to

this as the “general” dataset). In the MP method, the initial MP trees were performed with heuristic searches, starting trees determined by 10,000 random taxon addition, tree bisection-reconnection (TBR) branch swapping algorithm, gaps treated as missing data. Multiple character states in the same taxon were treated as substitution codons, and all characters treated as equally weighted. A strict consensus tree was computed in cases where multiple equally parsimonious trees were obtained. The consistency index (CI) and the retention index (RI) were also calculated in PAUP*10b. The topologies of MP trees were evaluated by both bootstrap (Felsenstein, 1985) and Bremer support (Bremer, 1988, 1994). Bootstrap analyses were performed with heuristic searches, 100 random taxon addition, TBR algorithm, and only parsimony-informative sites were used to increase variability. The bootstrap replicates were set to 1000. Bremer support (BS) was carried out using the program TreeRot.

v3 (Sorenson and Franzosa, 2007) in conjunction with PAUP*, and the number of heuristic searches with random-taxon addition for each branch was set to 100. In BI method, the software Modeltest v. 3.06 (Posada and Crandall, 1998) was used to determine the best fit model. Model selection was determined according to the hierarchical likelihood ratio test (hLRT).

The best fit model of GTR+Г+I [General Time Reversible (GTR), invariable site (I), and gamma distribution (Г)] were used. Six chains (five heated and one cold) were run for one million generations and sampled trees every 100 generations. The log-likelihood scores were plotted against generation time to determine when stationarity was reached. This process became stable before the first 2500-3000 trees, and therefore the first 3000 trees were treated as “burn-in” and the remaining

trees were used for representing the posterior probability. In BI, only one specimen of Niphanda fusca (Voucher ID: L1-2048) was set as outgroup due to the single-outgroup setting of Bayesian analysis.

Eliot (1973) in his classification admitted his failure to subdivide the huge tribe Polyommatini, but pointed out the similarity of the male genitalia of the Euchrysops and Polyommatus sections. In addition, according to the first run results (Figure 4), the analyses (MP and BI methods) identified that the Everes section was the closest group to Eliot’s Polyommatus section than to other Polyommatini sections.

Therefore, Everes argiades (Voucher ID: L1-270) and Euchrysops cnejus were chosen as functional outgroups, the remaining 57 terminals of the

Polyommatus-section as ingroup (here referred to as the “specific” dataset)

and all other taxa other than outgroups for the specific dataset were excluded from the analyses. MP was performed using the same settings as the first run. In addition, Partitioned Bremer support (PBS) was calculated for evaluating support and congruence among partitions. In BI, the best fit model for the specific dataset was set as the GTR+Г+I model based on Modeltest results, and subsequently the following settings used those of the first run. The first 3001 trees were treated as “burn-in” and the remaining trees were used to calculate posterior probabilities. ML similarly used the model of GTR+Г+I according to hLRT on this dataset, and the values of Г and I were set according to Modeltest suggestions (Г=0.66 and I=0.55). Tree topology search was set to SPR (Subtree Pruning and Regrafting). The initial tree was used default option (BIONJ), and nucleotide frequencies were based on “empirical” option, and the categories of discrete gamma model were set to four. Bootstrap analyses

based on 1000 pseudoreplicates were carried out to evaluate the support for all branching relationships.

For reporting the phylogenies, bootstrap support values ranging from 50-70% are referred to as “weakly supported”, those from 71-90% as

“moderately well supported”, and values higher than 90% are as

“strongly supported” (in accord with Silva-Brandão et al., 2008).

Bayesian posterior probability values from 0.5-0.75 are referred to as

“weakly supported”, those from 0.76-0.95 as “moderately well supported”, and those over 0.95 as “strongly supported”. Conflict between the three genes is detected by PBS values.

Ecological character mapping of hostplant associations

Evolutionary relationships between the butterflies and their hostplant families were investigated. Hostplant records were collected from several sources (Table 2). The optimized algorithms were based on parsimony, and character states were treated as unordered and equal weight by using Mesquite 2 (Maddison and Maddison, 2009). The characters were coded as binary (0 and 1) according to use or non-use of hostplant families, and missing data was coded as “?”. To detect whether there was phylogenetic signal in hostplant use, the methodology (modified PTP test) proposed by Wahlberg (2001) was used. I performed 300 random reshufflings of character states by using the program Mesquite 2. A significant phylogenetic signal is observed when P value is less than 0.05 (Faith and Cranston, 1991). In this study, the probability that the observed parsimony steps for that character do not differ from random is (n+1)/300, where n is the number of replications as short as or shorter than the

observed pattern.

II-3. Results

Characteristics of the dataset

The total alignment data set contained 3437 base pairs (bp), of which 1530bp was from mitochondrial COI, 682bp from mitochondrial COII, and 1225bp from nuclear EF-1α. No stop codon was found throughout the whole dataset, and only COII showed a 6-bp indel, which was caused by a two-codon insertion in Chilades trochylus (Voucher ID: L1-6100 and L1-2443) and a one-codon insertion in Plebejus argyrognomon (Voucher ID: L1-993) in the same region. There were 945 parsimony-informative sites found in the general dataset and 705 parsimonious sites in the specific dataset.

Phylogenetic relationships General dataset

The MP method over the whole combined dataset resulted in 16 most equally parsimonious trees (tree length=4950 steps, CI=0.338, RI=0.715).

The strict consensus tree showed that all the members within the tribe Polyommatini were strongly supported (Figure 4), whereas the relationships between Polyommatini sections were still unclear. The

Everes section is the closest to Polyommatus section among the sampled

sections, and all the Chilades butterflies are grouped into the

Polyommatus section. In the BI method, the dataset reached stationarity

before generation 300,000. I discarded the first 3,001 trees and computed the consensus tree from the remaining 14,000 trees. The phylogenetic tree has a similar topology with MP method. The monophyly of the tribe Polyommatini was well supported, and the relationships between the Polyommatini sections represent weak to moderate support. The Everes section also represent the closest to Polyommatus section as in the MP method, and Chilades butterflies represent two separated groups in the good support of the Polyommatus section (Figure 5).

Specific dataset

Parsimony searches resulted in 14 most parsimonious trees (tree length=2896 steps, CI=0.454, RI=0.751). The strict consensus tree shows that the Polyommatus section is well supported and contains four major groups (clade A-D, Figure 6). Clade A, including the genus Echinargus and Hemiargus, representing the Neotropical Polyommatus section shows both high bootstrap value and Bremer support (Table 11). Clade C which includes most of the Polyommatus genus-group proposed by Bálint and Johnson (1997, 1999) including Aricia, Plebejus, Albulina, and

Polyommatus has moderate bootstrap and Bremer supports. The genus Chilades is not included in the clade C, and this genus is divided into

another two clades instead. Clade B which includes most species of genus

Chilades, has moderate bootstrap and Bremer supports, while clade D,

including C. putli, C.minuscula, C. trochylus, and C. yunnanensis, has good bootstrap and Bremer supports. Clade D is more closely related to Clade C than to Clade B.

The three Chilades species-groups are mixed between clade B and D

and the genus is therefore polyphyletic. Group B contains all species-groups, with the galba-species group and lajus-species group in its basal part, and the cleotas-species group forming a subordinate clade of clade B. Clade D also belongs to the lajus-species group. The hitherto uncategorized Chilades species, C. yunnanensis, also belongs to the clade D, three of which (C. putli, C. minuscula and C. trochylus) were formerly included in Freyeria (Bascombe et al., 1999; Parsons, 1999; Ackery et al., 1995).

Bayesian analyses for the specific dataset also reached stationarity before generation 300,000. Again the first 3,001 trees were discarded and computed the consensus tree from the remaining 14,000 trees. The topology is similar to the parsimony consensus tree and four major clades are revealed. This time however, the major Chilades group (clade B) is more closely related to clade A, and the minor Chilades group (clade D) is more closely related to clade C. The log-likelihood from the ML method was -19380.86, and the resulting bootstrap values are labeled on Figure 6. ML shows a similar topology to the BI method. As before, four major clades with good support are revealed.

Evolution of hostplant associations

Tracing hostplant uses in the Chilades strict-consensus tree, the ancestral states of the tribe Polyommatini and the section Polyommatus are the family Fabaceae (Figure 8). The hostplant uses in Chilades butterflies (Clade B) are divided into two components: the basal lineages feed on rosid plants (including Fabaceae and Rutaceae) as other

Polyommatus-section butterflies, whereas the tip clade feeds on

Cycadaceae. In Freyeria butterflies (Clade D), although the hostplant use of the species C. yunnanensis is still unknown, the ancestral hostplant use is also the family Fabaceae. The modified PTP test used to detect phylogenetic signal was significant (P=0.003), implying that the distribution of Cycas-feeding trait among taxa is phylogenetically associated.

II-4. Discussion

Phylogenetic relationships of Chilades and Freyeria butterflies

The molecular data support the monophyly of Eliot’s Polyommatus section and Chilades butterflies as currently conceived are clearly polyphyletic. Bálint and Johon (1997, 1999) placed the genera Luthrodes,

Edales, Lachildes, and Freyeria as synonyms of the genus Chilades. The

results support the treatment of first three genera, but the genus Freyeria should be treated as an independent group because both clades are well supported (Figure 6, clade B and D). The genus Freyeria here was formally resurrected, including members of F. trochylus and F. minuscula,

F. putli, and F. yunnanensis, comb. nov. Although phylogenetic

relationships among the basal lineages of Polyommatus section are still unresolved, Freyeria (clade D) is more closely related to other members of the Polyommatus genus-group (clade C) than to the genus Chilades (clade B) (Figure 6). Moreover, three Chilades species-groups exhibit a mixed pattern, even when the members of Freyeria as conceived here are excluded. The cleotas species-group represent the terminal group in clade

B (Figure 6), however, this species-group and the galba species-group are the two members of clade B. If these species-groups are removed from clade B, the remaining taxa are rendered paraphyletic. C. yunnanensis is rare and only one female specimen is known in the Natural History Museum (BMNH). It has been recollected in the western Yunnan, where it inhabits mountainous areas.

Several new observations were made during the course of this study.

Firstly, the molecular data show that the Everes section are more closely related to the Polyommatus section, with good support (Figure 4), even though Eliot (1973) pointed out that the Euchrysops section have similar morphological characters in the aedeagus to the Polyommatus section.

Moreover, a hypothetical relationship proposed by Bálint and Johnson (1997) was that the Nabokovia genus-group is sister to the Polyommatus genus-group. The molecular data provide no support on this point, whereas the Nabokovia genus-group is evident as one of the basal lineages of the large section Polyommatus based on the combined molecular dataset (Figure 6). Moreover, the topology indicates that

Chilades butterflies are a group that originated out of Africa (similar

cases are represented by Kodandaramaiah and Wahlberg, 2007 and Aduse-Poku et al., 2009). African Chilades represent basal lineages of clade B, Central Asian Chilades intermediate lineages, and Asian species tip lineages. It is interesting to note that Freyeria represent the reverse pattern to Chilades butterflies, the Asian species represent in the basal part and African species at the tip clade.

Relationships among the Polyommatini sections

Although the main focus of this study was to examining the monophyly of Chilades butterflies, the relationships among the genera of the Polyommatus section and the sections among the tribe Polyommatini are worth discussing. Eliot’s great work (1973) built a basal classification of Lycaenidae, and many authors have followed his fundamental classification (Scoble, 1986; Bascombe et al., 1999; Braby, 2000).

Although Eliot (1973) admits himself a failure to find good characters for subdividing the very large tribe Polyommatini into a few major natural groups, the molecular data still strongly support the monophyly of the tribe Polyommatini (Figure 4). Bálint and Johnson pioneered the task of reclassifying Eliot’s Polyommatus section, based on male and female genitalic characters, with a focus on the aedeagus and female terminalia.

They provided valuable information about Neotropical blues using good taxon sampling within this section of lycaenids. However, their results indicated that the generic grouping still needs further clarification because the sampled species in the genera Aricia and Polyommatus seems not to compose monophyletic groups (Figures 6 and 7). More sampling efforts are needed to clarify the relationship in this large group.

Evolution of hostplant associations of Chilades butterflies

Chilades mindora, C. cleotas, and C. pandava, the three species that

are known to be specialized on Cycas plants form a monophyletic group within the clade of Chilades sensu stricto (Figure 8), suggesting the colonization of cycads occurred just once in the evolutionary history of these lycaenids. The phylogeny indicates that the cycad-association was

derived from a legume association, while the Rutaceae-association was an independent event unrelated to the colonization of cycads. The reasons that Chilades butterflies have shifted on to very divergent hostplants are still unknown. Pierce (1985) and Pierce et al. (2002) suggested that cycad-feeding behavior in Lycaenidae might have been facilitated by an enhanced requirement for nitrogen in larvae which can use for maintaining the interaction with ants. However, Fiedler (1995a, 1995b) questioned this point through comparing hostplant uses of tropical with temperate lycaenids. He also suggested that flavonoids might play a role for lycaenids choosing their hostplants (Fiedler, 1996). The identification of key innovations in the case of Chilades requires further study.

References

Ackery, P.R., Smith, C.R and Vane-Wright, R.I., 1995. Carcasson’s African butterflies:

an annotated catalogue of the Papilionoidea and Hesperioidea of the Afrotropical region. CSIRO Publishing, Melbourne, Australia. 816 pp.

Aduse-Poku, K., Vingerhoedt, E., Wahlberg, N., 2009. Out-of-Africa again: A phylogenetic hypothesis of the genus Charaxes (Lepidoptera: Nymphalidae) based on five gene regions. Mol. Phylogenet. Evol. 53, 463-478.

Allen, T.J., Brock, J.P., Glassberg, J., 2005. Caterpillars in the field and garden: a field guide to the butterfly caterpillars of North America. Oxford University Press, New York, USA. 240 pp.

Als, T.D., Vila, R., Kandul, N.P., Nash, D.R., Yen, S.H., Hsu, Y.F., Mignault, A.A., Boomsma, J.J., Pierce, N.E., 2004. The evolution of alternative parasitic life histories in large blue butterflies. Nature 432, 386-390.

Bálint, Z., Johnson, K.. 1997. Reformation of the Polyommatus section with a taxonomic and biogeographic overview (Lepidoptera, Lycaenidae, Polyommatini). Neue Entomol. Nachr. 40, 1-69.

Bálint, Z., Johnson, K.. 1999. Annotated list of type specimens of Polyommatus sensu Eliot of the Natural History Museum, London (Lepidoptera, Lycaenidae).

Neue Entomol. Nachr. 46, 1-89.

Bascombe, M.J., Bascombe, F.S., Johnston, G., 1999. The butterflies of Hong Kong.

Academic Press, London, UK. pp. 1-422.

Bayliss, J., Burrow, C., Martell, S., Staude, H., 2009. An ecological study of the relationship between two living fossils in Malawi: the Mulanje Tiger Moth (Callioratis grandis) and the Mulanje Cycad (Encephalartos gratus). African J.

Ecol. (in press)

Beccaloni, G.W., Viloria, A.L., Hall, S.K., Robinson, G.S., 2008. Catalogue of the hostplants of the Neotropical butterflies. Natural History Museum, London, UK. 536pp.

Bernays, E.A., Chapman, R.F., 1994. Host-plant selection by phytophagous insects.

Chapman & Hall, New York, USA. 312pp.

Boggs, C.L., Watt, W.B., Ehrlich, P.R., 2003. Butterflies: ecology and evolution

Boggs, C.L., Watt, W.B., Ehrlich, P.R., 2003. Butterflies: ecology and evolution

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