Euthalia ipona, Fruhstorfer, 1913

Yago, Masaya, Yokochi, Takashi, Kondo, Mariko, Braby, Michael F., Yahya, Bakhtiar, Peggie, Djunijanti, Wang, Min, Williams, Mark, Morita, Sadayuki & Ueshima, Rei, 2012, Revision of the Euthalia phemius complex (Lepidoptera: Nymphalidae) based on morphology and molecular analyses, Zoological Journal of the Linnean Society 164 (2), pp. 304-327 : 312-318

E. IPONA , AND E. EUPHEMIA

As shown in Figures 11–14 , three analyses (COI, ND5, and Tpi) showed high support for a clade corresponding to the E. phemius complex (BV 98–100%, PP 1.00), whereas EF-1a showed little support for this relationship (BV 56–66%, PP <0.90). Moreover, this group was always sister to the clade composed of E. aconthea , E. oriens , and E. alpheda , or the clade adding E. agnis and E. tinna to the three species. This sister-group relationship is also supported with some high values (BV <50–83%, PP 0.90–0.92), although very low values (BV <50%, PP <0.90) were obtained in the COI trees. In the E. phemius complex, not only were E. phemius , E. ipona , and intermediate forms represented as a polytomy with very small genetic distances (COI: 0.15–1.20%, ND5: 0.12– 0.84%, EF-1a: 0.18–0.35%, Tpi: 0.41–1.65%), but also some individuals shared identical sequences in both mitochondrial and nuclear genes. Judging from the genetic distance, the topologies, and the high frequency of gene flow, the results indicated that taxonomic re-examination of E. phemius and E. ipona was necessary.

Euthalia phemius was originally described as a species of genus Itanus (type locality: Silhet, Bangladesh; Doubleday, 1848), and the type male shown in the original description is deposited in the NHM. Euthalia ipona was first named as a subspecies of E. phemius (type locality: Perak, west Malaysia; Fruhstorfer, 1913), and one male type is also preserved in the NHM, although the total number in the type series was not noted in the original description. We also examined the wing markings and male genitalia of the two species, including the type specimens ( Figs 1–4 , 15–17 ). On the forewing upperside, E. phemius differs from E. ipona as follows: (1) in male, subapical white striae fine but clearly appearing in bases of cells 3–6 (the striae absent or obscure in E. ipona ); (2) in female, oblique median white band very broad and long, and extended from subdiscal portion of cell 1b or 2 to base of cell 6; often present also in base of cell 11 and/or apical portion of 12 (the band absent or vestigial in E. ipona ). Intermediate forms between the two species have shorter and narrower white striae (male) or white band (female), appearing only in bases of cells 4–6 ( Figs 5, 6 ). In the male genitalia we found that the two species, together with E. euphemia , share a unique character in that the ventral portion of the tegumen is produced into a small triangular projection, which is close to the base of the costa ( Figs 15–18 ). However, we could detect

EEu-Borneo4 1 1-2 3-4, HongKong 1-2, VietNam 1, Thailand 1-2, Myanmar 2 2 2, Laos 2, Myanmar 3, EIp-Thailand1-2 2 1 1, Malaysia 3-4, Myanmar 1, 4, India 1 3-4, EPhIp-Malaysia2 5, EIp-Malaysia2, 5 India 2 1, 3 1-2 1 -4 EAl-Malaysia1 5, EIp-Malaysia5 3 EOr-Indonesia1 4 EAc-Langkawi1 VietNam 1, Thailand 1-2 -3, Myanmar 2-4 1, Langkawi1-2, Malaysia 3-4 2 1 1 1 1 1 1 13 14 TPe-Malaysia1 0.01 0.1

almost no morphological differences between the two species or the intermediate forms in the male genitalia, although the two species showed small geographical and individual variations in the distal portion of the valva ( Figs 15–17 ). Variation of the male genitalia amongst races or subspecies in the Nymphalidae is often found in nonfunctional parts such as the dorsal and distal margins of the valvae, which do not make contact with the female genitalia during copulation ( Goulson, 1993). Additionally, there was no variation of the male genitalia between the two species in overlapping areas of their distributions ( Figs 16 , 17 ). The morphological and molecular evidence therefore indicates that E. phemius and E. ipona have not become reproductively isolated and that gene exchange is still taking place. Thus it would be reasonable to treat E. ipona as a form of E. phemius .

The monophyly of E. euphemia was strongly supported, with relatively high values ( Figs 11–14 ; BV 78–100%, PP 0.96–1.00). Although E. euphemia was regarded as the sister group to the remaining E. phemius complex in the COI and EF-1a trees ( Figs 11 , 13 ), the ND5 and Tpi trees showed that this species is internal within the clade composed of E. phemius and E. ipona ( Figs 12 , 14 ), so there was little phylogenetic resolution between E. phemius (and E. ipona ) and E. euphemia . Although there are some cases in which recently diverged species fail to form reciprocal monophyly in a locus of mtDNA (ex. Kandul et al., 2004; Oliver & Shapiro, 2007), a single mitochondrial locus generally becomes reciprocally monophyletic much faster than does a single nuclear locus ( Hudson & Coyne, 2002). Moreover, the time for reproductively isolated lineages to display reciprocal monophyly in a majority of nuclear loci can be considerably long ( Hudson & Coyne, 2002), so different nuclear loci will often conflict, especially in comparisons of closely related taxa ( Maddison, 2008). Therefore, it is not easy to decide clearly whether E. euphemia should be a species or a subspecies based on our molecular analyses. However, the genetic distances between E. euphemia and the remaining E. phemius complex species were rather small in all analysed genes (COI: 1.20–2.10%, ND5: 0.60–1.32%, EF-1a: 0.35–0.53%, Tpi: 0.82–2.06%), as compared to the genetic distances between the other Euthalia species (COI: 3.30–9.00%, ND5: 3.49–8.65%, EF-1a: 0.71–5.13%, Tpi: 3.30–13.58%). Nevertheless, as speciation some- times can be caused with low pairwise sequence divergence of mtDNA in Lycaenidae ( Kandul et al., 2004; Oliver & Shapiro, 2007), we can only say from our molecular analyses that the specific status of E. euphemia is vague. The results suggest that reproductive isolation may not be complete, or that barriers may have recently been established between E. euphemia and the remaining E. phemius complex.

Defining species is problematic, because there is no concrete concept free from any ambiguities. As de Queiroz (1998, 2005, 2007) hypothesizes, during the course of speciation, the two separating lineages acquire different properties relative to each other (species criteria), such as being phenetically distinguishable, reciprocal monophyly, and pre- and postzygotic reproductive isolation. The more evidence of lineage separation is available, the better one can say that different species exist. Therefore, in our case, more data, specifically morphological data, which we will show below, would be required to draw our conclusion on the specific status of E. euphemia .

Euthalia euphemia (type locality: Kina Baru, Borneo) has been treated as a valid species by some researchers since its description by Staudinger (1896), although Corbet (1945), D’Abrera (1985), and Yokochi (1999) ranked it as a subspecies of E. phemius . Tsukada (1991) tentatively placed the Bornean population as a separate species, but pointed out that there is a possibility that it should be regarded as a subspecies of E. phemius . Tsukada also illustrated the male genitalia of E. euphemia , but did not compare them with those of E. phemius . We investigated the wing markings and male genitalia of E. euphemia , including the type deposited in the ZMHU ( Figs 7, 8 , 18 ), and compared them with those of E. phemius from every locality ( Figs 1–6 , 15–17 ). We found that E. euphemia differs from E. phemius in the following wing characters ( Table 2): (1) both sexes generally somewhat smaller; (2) wing shape tending to be slightly rounded in both sexes, so that anal angles of the fore- and hindwings are usually slightly wider; (3) in male, submarginal blue band on hindwing underside pale violet (pale blue or pale green in E. phemius ); (4) female hindwing upperside with wide submarginal pale purple band; (5) oblique median white band on forewing upperside of female bifurcate. H, height; L, length; ML, maximum length.

Mean ± standard deviation of measured values is in square brackets. The anal angles of fore- and hindwings were measured as the angle formed between the inner margin and the line connecting tips of veins 1a and 1b.

Venation system follows Tsukada (1991).

The important point to note is that Euthalia species generally show specific differences in the male genitalia, especially the shape of the valva ( Tsukada, 1991). Tsukada did not report the male genital structures of E. phemius , and did not compare this to E. euphemia . We have for the first time compared in detail the genital structures for both E. phemius and E. euphemia . In particular, the shapes of valvae were analysed based on the EFDs ( Table 3; Fig. 19 ; see also Material and methods). As a result, there were no differences in not only the male genital valvae but also the other genitalic parts between E. phemius (and E. ipona ) and E. euphemia ( Figs 15–19 ; Table 2).

Based on the genetic analyses and genitalic differences, we consider E. euphemia to be a subspecies of E. phemius . Probably, the small genetic variation, and the characteristic wing markings, of E. p. euphemia were caused by a bottleneck effect (founder effect) as a result of promotion of genetic drift, i.e. the ancestral E. p. euphemia had been isolated to a confined area such as Borneo, where a loss of genetic variation rapidly occurred and in a short period the new population was distinctly different, both genetically and phenotypically, with an accumulation of genetic mutations. This taxonomic statement may be clarified through breeding experiments planned in the near future.

In this study we also analysed divergence times in the E. phemius complex from combined sequences (1497 bp) of mitochondrial COI and ND5 genes. There are two reasons why mitochondrial sequences in butterflies are particularly useful for estimating genetic divergence both within and between species ( Mallet et al., 2007). First, recombination between mitochondria, because of unisexual inheritance, is unlikely to occur, and thus genetic divergence is considered not to be influenced by occasional introgression. Secondly, in butterflies, hybrid females are often sterile or have low viability according to Haldane’s rule ( Coyne, 1985; Presgraves, 2002; Lukhtanov et al., 2005). Haldane’s rule implies that introgression of maternally inherited mitochondria is prevented at an earlier stage of speciation than that for nuclear loci in female-heterogametic species ( Sperling, 1990; Jiggins et al., 2001a, b; Naisbit et al., 2002), which may be transferred between species by backcrossing of male hybrids. Using this combined mitochondrial sequence for divergence time estimation, a linearized NJ tree based on Kimura’s twoparameter model was constructed (Fig. 20). Analysis based on Tajima’s relative-rate test confirmed that a molecular clock could be hypothesized for our data set (P <0.05). A molecular clock of 1.1–1.2 ¥ 10 -8 substitutions per site per year ( Brower, 1994) was applied (see Material and Methods). Judging from the linearized tree, the common ancestor of Euthalia was initially divided into six lineages, and this diversification occurred about 2.5–3.1 Mya. Beginning around 3 Mya, fluctuations between glacial and interglacial periods gradually occurred throughout the world ( Lisiecki & Raymo, 2005; Reymo, Lisiecki & Nisancioglu, 2006). The start of the extreme climatic variation almost corresponds to early divergences within the genus. After this, the common ancestor of the E. phemius complex appeared about 2.1–2.3 Mya. In the lineage of the E. phemius complex, the divergence age of the ancestral E. phemius phemius and the ancestral E. phemius euphemia was estimated at about 0.5–0.6 Mya. We infer that at this point in time E. phemius was divided into two populations by the formation of the Strait of Malacca between Borneo and the Malay Peninsula, extending from Indochina, perhaps because of a climatic or geographical change. Subsequently, it is considered that one lineage evolved into the extant E. phemius phemius , widely distributed in the Asian continent, whereas the other was isolated in a limited area for a long period and became the extant E. phemius euphemia , endemic to Borneo. Euthalia monina may also have evolved in a similar way, the nominotypical subspecies occurring on the Malay Peninsula and the subspecies E. bipunctata (Snellen van Vollenhoven, 1862) on Borneo ( Tsukada, 1991; Eliot, 1992). The division of the populations of E. monina seems to be at almost the same time as those of E. phemius (Fig. 20).

Figures

Figure 19. Shape variation amongst male genital valvae of Euthalia species (A–P). The first (PC1) and second (PC2) principal components are plotted. A: Euthalia phemius (N = 10), B: Euthalia ipona (N = 5), C: Euthalia euphemia (N = 2), D: intermediate form (N = 1), E: Euthalia aconthea (N = 2), F: Euthalia alpheda (N = 2), G: Euthalia agnis (N = 1), H: Euthalia tinna (N = 1), I: Euthalia oriens (N = 2), J: Euthalia mahadeva (N = 2), K: Euthalia monina (N = 2), L: Euthalia eriphylae (N = 2), M: Euthalia merta (N = 2), N: Euthalia lusiada (N = 2), O: Euthalia anosia (N = 2), P: Euthalia lubentina (N = 2).

Figures 11–12. Phylogenies of the Euthalia aconthea group including the Euthalia phemius complex. The topologies show Bayesian inference trees based on mitochondrial cytochrome c oxidase subunit I (COI; 11) and NADH dehydrogenase subunit 5 (ND5; 12) genes using the program MrBayes 3.1.2. Identical sequences were treated as a single operational taxonomic unit (OTU). Numbers indicate bootstrap values from neighbour-joining (top left) and maximum parsimony (top right) analyses, and posterior probabilities from Bayesian analysis (bottom). Only bootstrap values> 50% and Bayesian posterior probabilities> 0.50 are shown. Branch lengths represent nucleotide substitutions per site. The abbreviations of OTUs correspond to those in Table 1.

Figures 13–14. Phylogenies of the Euthalia aconthea group including the Euthalia phemius complex. The topologies show Bayesian inference trees based on nuclear Elongation factor-1 alpha (EF-1a; 13) and triose-phosphate isomerase (Tpi; 14) genes using the program MrBayes 3.1.2. Identical sequences were treated as a single operational taxonomic unit (OTU). Numbers indicate bootstrap values from neighbour-joining (top left) and maximum parsimony (top right) analyses, and posterior probabilities from Bayesian analysis (bottom). Only bootstrap values> 50% and Bayesian posterior probabilities> 0.50 are shown. Branch lengths represent nucleotide substitutions per site. The abbreviations of OTUs correspond to those in Table 1.

Figures 1–8. Three species of the Euthalia phemius complex (A: upperside; B: underside). The specific classification follows Tsukada (1991) (See Material and methods in text). 1, Euthalia phemius (Doubleday, 1848), syntype ♂, Silhet, Bangladesh, labelled as ‘Type ♂ Itanus phemius Doubleday/ Silhet (undecipherable characters) 46–31/ B.M. TYPE No. Rh10128 Adolias phemius ♂ Doubleday’ (NHM) (©NHM); 2, ditto, ♀, Naungmon, Kachin, Myanmar; 3, Euthalia ipona Fruhstorfer, 1913, syntype ♂, Perak, W. Malaysia, labelled as ‘Cunong Ijan’ (NHM) (©NHM); 4, ditto, ♀, Mt. Muan Chone, Ranong, Thailand; 5, intermediate form (A) between E. phemius and E. ipona, ♂, Cameron Highlands, Pahang, Malaysia; 6, intermediate form (B) between E. phemius and E. ipona, ♀, Bukit Bintang, Terengganu, Malaysia; 7, Euthalia euphemia Staudinger, 1896, lectotype ♂, labelled as ‘Origin. (red)/ Euphemia Stgr./ Kina Balu 12–1500 m. N. Borneo 1894 Waterstradt/ Lectotype ♂, Euthalia euphemia Staudinger, 1896. Designated by T. Yokochi, 1997’ (ZMHU); 8, ditto, ♀, Tambunan Rd., Crocker Range, Sabah, Borneo, Malaysia.

Figures 15, 16. Male genitalia of the Euthalia phemius complex (without left valva, lateral views). 15, Euthalia phemius, Khasi Hills, Meghalaya, India (EPh-India3); 16, E. phemius, Cameron Highlands, Pahang, Malaysia (EPh-Malaysia6). Scale bar = 1 mm.

Figures 17, 18. Male genitalia of the Euthalia phemius complex (without left valva, lateral views). The specific classification follows Tsukada (1991) and Eliot (1992). 17, Euthalia ipona, Cameron Highlands, Pahang, Malaysia (EIp-Malaysia6); 18, E. euphemia, Pontianak, W. Kalimantan, Borneo (EEu-Borneo2), Indonesia. Scale bar = 1 mm.