Cleorodes Warren , 1894 does not belong in the tribe Boarmiini ( Lepidoptera : Geometridae )

The geometrid genus Cleorodes is shown to belong in the tribe Gnophini (sensu lato) and not in Boarmiini as previously assumed. The conclusion is based on an analysis of morphological characters of a number of genera in these tribes. Moreover, the result is unambiguously supported by a phylogenetic analysis of DNA sequence variation in three nuclear gene regions (segments D1 and D2 of 28S rRNA, and elongation factor 1 ) and a mitochondrial gene, cytochrome oxidase-1. The phylogenetic hypothesis is based on a combined sequence data set, which was analysed using direct optimisation. 303 * Corresponding author; e-mail: toomas.tammaru@ut.ee miini, and indicate affinities with the tribe Gnophini in the broad sense. This is supported by a rigorous phylogenetic analysis of DNA sequences. It is concluded that Cleorodes is a gnophine, not a boarmiine genus as traditionally assumed. MATERIAL AND METHODS


INTRODUCTION
The largest subfamily of Geometridae, the Ennominae, with over 9700 described species (about 45% of all Geometridae: Minet & Scoble, 1999), is well defined by a morphological character, the weakening or absence of the vein M2 in the hind wings.On the other hand, there is no consensus about the taxonomic affinities of the numerous currently recognized tribes within the subfamily.Nevertheless, with few exceptions, these can be divided into two major branches on the basis of a pupal character, the structure of the cremaster (Forbes, 1948;Holloway, 1994).In particular (Fig. 1A, B), the cremaster may either (1) end in two separate stout (D2) setae, associated with three pairs of thin, hooked side setae (D1; Sd1; L1)defining the informal "ennomine group" of tribes -, or (2) lack the side setae (Fig. 1C, D) and end in a cone-or stick-shaped, apically bifid projection, a condition characteristic of the "boarmiine group" of tribes (compare also text figures 119 and 122 in Hausmann, 2001).It is proposed that the former, i.e. the combination of 2 stout + 6 side setae is ancestral for Ennominae, and the loss of the side setae in e.g.Macariini, Bistonini and Boarmiini represents a derived condition (Holloway, 1994).
Within the group with a bifid cremaster, a high number (131 species listed by Prout 1912Prout -1916) ) of geometrid moths were initially grouped within the genus Boarmia Treitschke, 1825.Later, Wehrli (1939-53) subdivided the large Boarmia into numerous subgenera.More recently, the subgenera were raised to the rank of genera (Herbulot, 1961(Herbulot, -1962;;Leraut, 1997), and considered to collectively form the tribe Boarmiini within the subfamily Ennominae.These moths are relatively uniform in appearance, with the wing pattern resembling tree bark, and have traditionally been viewed as an uncontroversial natural unit.Some differently looking genera have been added to the tribe (e.g.Arichanna, by Herbulot, 1962), but the systematic position of any of the "classical Boarmias" has never been questioned, neither has the tribe as a whole been subjected to a systematic taxonomic revision.
As a particular example, Cleorodes Warren, 1894 is treated as a separate genus within the Boarmiini group by all recent authors, e.g.Müller (1996), Viidalepp (1996), Leraut (1997) and Scoble (1999).The genus, with its single, lichenivorous (e.g.Pöykkö, 2006) European species C. lichenaria Hufnagel, 1767, was considered to be monotypic for a long time.Quite recently, however, another species, C. incerta Rungs, 1975 was described from Morocco.To our knowledge, no one has ever raised doubts concerning the taxonomic affinities of the genus.Consistently, Pato ka & Tur áni (2005), in their monograph on the pupal morphology of European Lepidoptera, group Cleorodes in the Boarmiini.Notably, however, in this key, this genus is reached between geometrids representing the ennomine group of tribes; i.e. as compared to the rest of the traditional Boarmias, it appears on the other side of the major morphological watershed subdividing the subfamily.Pato ka & Tur áni (2005) did not, however, comment on this situation.
In this paper, we show that C. lichenaria, in addition to an ennomine cremaster in the pupal stage (Fig. 1A), possesses also a number of other morphological traits that clearly distinguish it from typical representatives of Boar-miini, and indicate affinities with the tribe Gnophini in the broad sense.This is supported by a rigorous phylogenetic analysis of DNA sequences.It is concluded that Cleorodes is a gnophine, not a boarmiine genus as traditionally assumed.

Morphological study
Along with C. lichenaria, 64 Palaearctic species representing 37 genera of the geometrid subfamily Ennominae (Appendix) were examined with the aim to find and/or confirm distinctive characters of the tribes Boarmiini, and the closely related Bistonini, on the one hand; and Gnophini, on the other.The concept of Gnophini used in this study is a broad one (sensu Herbulot, 1961-62, including e.g. Aspitates, Dyscia and Siona).The still unsolved question of delimitation of the tribes included in the ever more broad concept of the Boarmiini by Holloway (i.e.Gnophini, Boarmiini and Bistonini) requires a major revision of respective genera and is beyond the scope of the present paper.
For the morphological study, moths from the collection of the Estonian University of Life Sciences, Tartu, Estonia, were used.Both female and male genitalia, as well as pupal exuviae, were examined when suitable material was available.Examination of genitalia followed procedures described by Hardwick (1950).Pupae were photographed with a Canon 350D.The digital images were enhanced using Adobe Photoshop.

Molecular analysis
In addition to C. lichenaria, two representatives of Boarmiini, Bistonini and Gnophini (sensu lato, see above) were included in the phylogenetic analysis of DNA sequences.A species of Ennomos was added as a typical representative of the informal group of the "ennomine tribes".To root the tree, two species from the subfamilies Archiearinae and Larentiinae were also considered, the latter being defined as an outgroup.The taxa used, along with where the specimens were collected, and Gen-Bank numbers of the sequences, are listed in Table 1.To exclude the possibility of a technical mistake, another specimen of C. lichenaria, from the same location, was examined and found to have identical sequences in particular gene regions.
Using a DNeasy Tissue Kit (Qiagen, Hilden, Germany), genomic DNA was extracted from freshly captured moths which were dried, frozen, or preserved in alcohol.Only parts of the specimens were used for DNA extraction, which allowed the voucher specimens to be preserved.For the phylogenetic analysis, the portions of two nuclear genes, D1 and D2 segments of the 28S rRNA gene and the elongation factor 1 (EF-1 ) gene, and a portion of a mitochondrial gene, cytochrome oxidase-1 (CO1) gene, were sequenced.PCR reactions were carried out with 5-100 ng of DNA template, 0.1 µM of each primer, 200 µM of each dNTP, 1 U of DyNAzyme II DNA polymerase (Finnzymes Oy) in 1 × DyNAzyme buffer supplemented with 1.5 mM MgCl2 in a PTC-100 Peltier Thermal Cycler (MJ Research Inc.) or in a Eppendorf Mastercycler gradient (Perkin-Elmer Corp.).

Phylogenetic analysis of molecular data
The sequencing of the three nuclear and one mitochondrial gene fragments produced an average of 2326 bp sequence data for phylogenetic analysis.The obtained sequences for all gene fragments varied in length due to the PCR amplification and sequencing techniques (e.g.primer combinations used, ambiguities in sequence ends), whereas only 28S D1 and D2 fragments were of variable length due to indels.The average lengths of the gene partitions were as follows: 285 bp for 28S D1, 441 bp for 28S D2, 959 bp for EF-1 , and 641 bp for CO1.The incongruence length difference test (Farris et al., 1994), which was used to test for the combinability of the different gene partitions, showed no significant incongruity between data sets (P > 0.9).The PCR amplification of the 28S D2 sequence of E. defoliaria was unsuccessful, and thus the molecular data used lack this sequence.

RESULTS AND DISCUSSION
Our morphological analysis of Palaearctic taxa showed that the tribes Boarmiini and Bistonini (sensu Herbulot, 1961Herbulot, -1962) ) share several well-defined characters that distinguish them from the representatives of the informal "ennomine group" of tribes (Table 2), and each have some of their own synapomorphies.In addition to the distinctive, and presumably derived, pupal character -the loss of side setae and fusion of D2 setae on cremaster, the modification of male abdominal sternite A3 deserves attention as a taxonomically promising synapomorphy.The question about the universal validity of these diagnostic traits cannot, however, be answered prior to a comprehensive phylogenetic study of the world fauna.Holloway (1994: p. 167) combined all the genera with the derived cremaster condition, shared possession of a fovea and of a transverse comb on the sternite A3 in males, in the Boarmiini sensu lato.However, earlier Pierce (1914) distinguished between the Boarmiini (characterized by spinose valva) and Bistonini (valva smooth), and this distinction was adopted in by the majority of subsequent taxonomic works (Herbulot, 1961(Herbulot, -1962;;Leraut, 1997).The distinction is well supported morphologically (Viidalepp, 1989).In particular, in Bistonini, pupae are characterized by angulate lateral projections on a stout cremaster, and by the absence of labium and fore femur (Pato ka, 1986).Bistonine moths, so far studied by the authors, (1) lack a fovea at the base of the male forewing, and (2) the setal comb ventrally on sternite A3 is lacking or replaced by a tuft of white hairscales.Bistonine taxa also have (3) uncus often ventrally bi-pointed and (4) valva in male genitalia usually simple, parallel-sided with strong costal region and (5) lack a harpe.Female genitalia are often characterized by (6) very long apophyses with oral tips flattened.Of the genera examined in this study, the following six share the listed "bistonine" characters, or most of them: Apocheima, Biston, Erannis, Larerannis, Lycia, and Phigalia.However, Apocheima and Erannis are exceptional in the presence of a thorny harpe on centre of valva in males.

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Uncus or gnathos often reduced, or gnathos longer than uncus, jaw-shaped Gnathos reduced Gnathos as strong as uncus and provided with cochlear (its medial projection) 5 Fovea usually lacking in male forewings Fovea lacking in males Male forewing with fovea present (Boarmiini), or absent (Bistonini) 4 Often pectinated to the apical segments Pectinated to apical segments Male antennae, if pectinated, then with apical segments filiform 3 Male abdominal sternite A3 without a comb of stiff setae Male abdominal sternite A3 with a transverse comb of setae (Boarmiini), or hair lock (Bistonini) 2 Cremaster with setae D1, SD1, L1 and D2 present, setae D2 separated Pupa: cremaster setae D1, SD1 and L1 reduced, D2 fused into a stem 1 Gnophini Cleorodes Bistonini + Boarmiini TABLE 2. Discriminative characters for the genus Cleorodes, and putative related groups.The table is based on the examination of a sample of Palearctic genera (see Appendix).
The genera of Boarmiini s. str.examined have a stickshaped, apically bifid projection on the cremaster which lacks lateral angulation.These moths typically also possess (1) well delimited harpe and cucullus on male valva, (2) a setal comb on sternite A3, and (3) a deep fovea on male forewing (with rare exceptions).Uncus is triangular with a short, finger-like distal projection, the gnathos is usually well sclerotised and provided with a strong, flat medial cochlear.In only one case, the ovipositor is telescopic with the apophyses longer than usual (Ectropis).The following seven genera share the "boarmiine sensu stricto" characters: Aethalura, Alcis, Cleora, Deileptenia, Ectropis, Paradarisa, Peribatodes.Holloway (1994) did not investigate the Gnophini in detail but included the group within his broadly defined Boarmiini.Morphological evidence, however, appears not to conform with this suggestion.Above all else, gnophine moths (with the exception of Gnophos itself, which differs in having a bicornute cremaster) possess the diagnostic pupal character, i.e. the distinctive combination of setae on cremaster (Pato ka & Tur áni, 2005; Fig. 1A,  B), which indicates that they belong to the "ennomine" group of tribes.Gnophini (sensu Herbulot, 1962) are characterized by a combination of the following characters: (1) the uncus short and birdhead-shaped; (2) the absence of a fovea on forewings; and (3) the absence of a comb of setae on abdominal sternite A3 in males; (4) a costal projection on valva usually present, bearing a spine or group of spines; (5) the juxta is often H-shaped with short basal and longer distal appendages.Of the genera studied: Acrognophos, Arbognophos, Aspitates, Bizia, Chariaspilates, Charissa, Chelegnophos, Cnestrognophos, Costignophos, Ctenognophos, Dyscia, Euchrognophos, Napuca, Nychiodes, Odontognophos, Dicrognophos, Dysgnophos, Elophos, Kemtrognophos, Pterygnophos, Rhipignophos, Rhopalognophos, Zystrognophos and, notably, Cleorodes, share the gnophine characters listed above.There is no doubt that C. lichenaria exhibits the "gnophine" spectrum of morphological char-acters (Table 2).Besides the distinctive cremaster condition (Fig. 1A), the short, convex, bird-head-shaped uncus provides perhaps the most convincing evidence.Cleorodes appears thus not to belong to the boarmiine branch of the subfamily Ennominae.
Consistently, the respective morphological parsimony analysis yielded a robust, fully resolved most parsimonious tree (to be presented elsewhere: Viidalepp, in prep.).The gnophine genera -including Cleorodeswere supported by five strict synapomorphies (listed in the previous paragraph), and were placed basal to the boarmiine and bistonine lineages.Both of these were supported by strict synapomorphies of their own (for discussion of bistonine and boarmiine characters, see also Viidalepp, 1989).
The results of the phylogenetic analysis of the molecular data unambiguously support the conclusions of the morphological study.In particular, the direct optimisation procedure produced a tree with two well-supported clades within the Ennominae (Fig. 2), which correspond to the major morphology-based subdivision of the subfamily into two groups.One of the clades was formed by representatives of the tribes Boarmiini s. str.(H.punctinalis and P. secundaria) and Bistonini (B.betularia and E. defoliaria).The results are consistent with the morphology-based idea that the Bistonini are monophyletic (Table 2) but hint at the possibility that the Boarmiini s. str.may be paraphyletic.If this is the case, one may prefer a broader concept of the Boarmiini which subsumes the Bistonini.Obviously, however, a much wider taxon sampling is required to resolve this question.
On the other hand, the two members of the Gnophini s. lat.(S. lineata and K. ambiguata) clustered together and formed a monophyletic group, which also included C. lichenaria.The Gnophini grouped together with Ennomos, confirming the morphology-based (Pato ka & Tur áni, 2005) view that the genera of Gnophini, sensu Herbulot (1961Herbulot ( -1962)), belong to the ennomine branch of the subfamily (cf.Holloway, 1994).
In summary, both the morphological and molecular evidence suggest that the position of Cleorodes in the Ennominae should be reconsidered.The genus should be removed from the tribe Boarmiini, and preliminarily placed in the Gnophini.Establishing the relationships of Cleorodes within the latter group will, however, require a major revision of respective genera.Such a revision is urgently needed not only because of the disputed status of Aspitatini (Holloway, 1994) but also because the nominate genus of the tribe, Gnophos, appears not to share all of the putative morphological synapomorphies with the rest of "Gnophini".Fig. 2. The maximum parsimony tree of eight geometrid species from the subfamily Ennominae.The tree is based on the combined sequence data of three nuclear and one mitochondrial gene fragment, analysed via direct optimisation in the program POY.The tree was rooted to Larentiinae and Archiearinae species."Gnophini" is used in its broad sense (Herbulot, 1961-62).Numbers above the branches indicate bootstrap support values.

TABLE 1 .
Information on the geometrids used in the molecular analyses.Collection site (EST -Estonia, FIN -Finland, SWE -Sweden) and year, collector's name, and GenBank accession numbers of sequences are indicated.