Allozyme Analyses of Two Closely Related Species of Eurema Butterflies (Lepidoptera: Pieridae) That Cannot Be Identified With Mitochondrial DNA Sequences

Allozyme Analyses of Two Closely Related Species of Eurema Butterflies (Lepidoptera: Pieridae)... Abstract The Japanese fauna of the yellow butterfly Eurema hecabe (L.) (Lepidoptera; Pieridae) populations in Ryukyu area (Southwestern Islands) were shown to differ from their Mainlands population (Honshu) in its seasonal forms of adult wing color pattern expression and in larval food habits. Recently, E. hecabe was divided into two species, E. hecabe and Eurema mandarina (de l’Orza) (Lepidoptera: Pieridae), based on their morphological, behavioral and genetic characteristics. However, whereas these two species are classified into two distinct groups based on nuclear DNA, both species are mixed in the phylogenetic tree from their mitochondrial DNA (mtDNA) sequences. The reason is a selective sweep by Wolbachia infection, and they can not be distinguished by DNA barcode method using mtDNA. We analyzed allozyme variation as a nuclear gene phenotype in 15 loci from 11 enzymes of the two closely related butterflies. Examining the esterase zymogram of each population, appearance frequency of Est-2 locus in each population varied greatly. E. mandarina showed the bands but E. hecabe showed no bands in Est-2 locus. The UPGMA and NJ phylogenetic trees branched out into two species—E. hecabe and E. mandarina—although their habitat overlapped in Okinawajima Is. populations. The results showed that the allozyme analyses were not affected by the selective sweep of Wolbachia infection between these two species. Geographic distribution of a natural population has historical and ecological components (Futuyma 1998). Genetic and ecological studies will provide available data for understanding how a single population in the past may have evolved into different populations within the same species or belong to closely related species widely distributed through different climatic zones. Eurema hecabe (L.) (Lepidoptera; Pieridae) is widely distributed from the tropical through subtropical to temperate regions in the Old world (Yata 1989). This species is a multivoltine butterfly and shows seasonal polyphenism (Summer/Wet morph and Autumn/Dry morph) in wing color pattern, which is associated with the reproductive diapause. Yata (1989) reported that populations of E. hecabe distributed in East Asia, including the Japanese population, belong to a single group on the basis of morphological characteristics, although the wing color pattern varies clinally with latitude. However, in Japan, which is the northern limit of the distribution area of E. hecabe, this species consists of two geographically separated types differing in fringe color (brown or yellow) of the upperside forewing (Kato 1999). These types are easily discriminated in the wing pattern of the autumn morph, although that of the summer morph is similar in appearance (Kato and Sano 1987, Kato and Handa 1992). Use of the host plants by larvae was also shown to differ between the two types (Kato et al. 1992). The brown type inhabits the subtropics of the Ryukyu Islands, and the yellow type inhabits mainly the temperate region including the Mainlands (e.g., Honshu) of Japan. Kato (1999) found that the butterflies of these two types sympatrically inhabit the subtropical Okinawa Island, which is in the northern part of the Ryukyu Islands. Their distribution on this island basically depends on the locality of their host-plant species. Laboratory experiments showed that females of each type produced offspring with the same fringe color and seasonal wing morph as their parents, and the ability for host utilization differed between them on this island. Therefore, it seems likely that there are two distinct populations of E. hecabe. Because of differences mentioned above, the two types of E. hecabe were judged to be of two different species, and the yellow type was named Eurema mandarina (de l’Orza) (Lepidoptera: Pieridae) (Shirouzu 2006). In the molecular phylogenetic analysis, these two species can be divided into two groups in the nucleotide sequence of nuclear DNA, but in the case of mitochondrial DNA (mtDNA), the two species were mixed in the phylogenetic tree and could not be clearly divided into two groups (Narita et al. 2006). It was revealed that mismatch of systematic relationship was caused by selective sweep by Wolbachia infection (Narita et al. 2006). For this reason, identifying these two species using mtDNA sequence is impossible because they have almost the same mtDNA sequence, and the two species can not be distinguished when using the DNA barcoding region (Hebert et al. 2003). Also, since nuclear DNA has heterozygous sequences from male and female chromosome, sometimes it requires an extra process: cloning. In addition, identification of the two Eurema butterflies in sympatric distribution areas is quite difficult using old specimens. Therefore, we attempted to classify the two groups by gene frequency analysis of allozymes, which is a traditional molecular phylogenetic method. To study the genetic variation and phylogeny of biological organisms including insects, a biochemical or molecular approach has been accepted as useful tools. Analysis of allozyme variation is also one of them used for clarifying genetic differences among populations within a species and among closely related species (e.g., Avise 1975, 1993; Ayala 1975; Jacobson et al. 1981; Pashley 1983; Nomura 1998). In the present study, we analyzed allozyme variation for the sympatric and allopatric populations of this species, by means of the polycarylamide gel electrophoretic technique for clarifying the genetic relationships among populations of E. hecabe and E. mandarina in Japan. Materials and Methods Insects Adult males and females of E. hecabe and E. mandarina were caught from 1991 to 1996 at different locations (Mainlands and Ryukyu [Southwest] Islands) in Japan (Table 1), and the identified species were recorded primarily by the fringe color (yellow or brown) of the upperside forewing. As an outgroup species for phylogenetic analyses, Eurema blanda (Boisduval) (Lepidoptera: Pieridae) was used, which were caught in Ishigaki Is. Then, the individuals that were frozen and stored at -80°C were used for analysis. We used the old samples for the experiment, because it is almost impossible to collect E. mandarina population in Rykyu Islands as a result of recent urban development projects. Table 1. Populations used and localities of collection in this study Species population  Locality  Latitude: longitude  Date  E. mandarina   Tokyo  Mitaka  N 35°41′: E 139°46′  Aug., 1991–1993   Shikoku  Kochi  N 33°32′: E 133°28′  July–Nov., 1993   Kyushu  Fukuoka  N 33°37′: E 130°25′  Sept., 1992   Oki-Em  Okinawa-jima  N 26°17′: E 127°47′  Oct., 1992, 1993  E. hecabe   Oki-Eh  Okinawa-jima  N 26°17′: E 127°47′  Mar.–Oct., 1991–1994   Ishigaki  Ishigaki-jima  N 24°20′: E 124°09′  Mar.–Oct., 1991–1992   Iriomote  Iriomote-jima  N 24°17′: E 123°51′  May, 1996  E. blanda (outgroup)   Ishigaki  Ishigaki-jima  N 24°20′: E 124°09′  May, 1996  Species population  Locality  Latitude: longitude  Date  E. mandarina   Tokyo  Mitaka  N 35°41′: E 139°46′  Aug., 1991–1993   Shikoku  Kochi  N 33°32′: E 133°28′  July–Nov., 1993   Kyushu  Fukuoka  N 33°37′: E 130°25′  Sept., 1992   Oki-Em  Okinawa-jima  N 26°17′: E 127°47′  Oct., 1992, 1993  E. hecabe   Oki-Eh  Okinawa-jima  N 26°17′: E 127°47′  Mar.–Oct., 1991–1994   Ishigaki  Ishigaki-jima  N 24°20′: E 124°09′  Mar.–Oct., 1991–1992   Iriomote  Iriomote-jima  N 24°17′: E 123°51′  May, 1996  E. blanda (outgroup)   Ishigaki  Ishigaki-jima  N 24°20′: E 124°09′  May, 1996  View Large An individual thorax (without wings and legs) was excised, and homogenized with 0.3 ml of 0.01 M phosphate buffer (pH 7.0) in a 1.5 ml disposable microcentrifuge tube and pellet pestle. The homogenates were centrifuged at 10,000×g for 20 min. under 4°C. The supernatants were stored at −40°C until electrophoresis. Electrophoresis and Analyses Electrophoresis was performed on a 7.5% polyacrylamide gel by the method of Davis (1964) using a vertical slab gel apparatus (Model AE6200: ATTO Corp., Tokyo) as described previously (Nomura and Ichinose 1990). After electrophoresis, the gels were stained for 11 enzymes as follows: esterases (EST, EC 3.1.1.-), glucose-6-phoshate dehydrogenase (G6PD, EC 1.1.1.49), glutamate oxaloacetate transaminase (GOT, EC 2.6.1.1), α-glycerophosphate dehydrogenase (α-GPDH, EC 1.1.1.8), hexokinase (HK, EC 2.7.1.1), isocitrate dehydrogenase (ICDH, EC 1.1.1.42), leucine amino peptidase (LAP, EC 3.4.11.1), malic dehydrogenase (MDH, EC 1.1.1.37), malic enzyme (ME, EC 1.1.1.40), phosphoglucomutase (PGM, EC 2.7.5.1) and 6-phoshogluconate dehydrogenase (6PGDH, EC 1.1.1.44). The staining methods used for all enzymes were those of Steiner and Joslyn (1979) with some modifications, except the staining methods for ME and EST, which were those of Loxdale et al. (1983) and Nomura and Ichinose (1990), respectively, with some modifications. We obtained zymograms of electrophoretic gels of 11 enzymes mentioned above, and estimated bands that were controlled by single locus. From our preliminary study, the appearance of bands of 11 enzymes on the gel did not differ between sexes and among ages of butterflies. We obtained 15 loci from the 11 enzymes (Table 2) and calculated the allele frequencies of 15 loci from electrophoretic data, and analyzed with POPTREE2 (Takezaki et al. 2010) to obtain Genetic Distance measures and the UPGMA (Unweighted pair strain method of arithmetic average; Sneath and Sokal 1973) and NJ (Neighbor-Joining) phylogenetic trees (Saitou and Nei 1987). Table 2. Allozyme frequencies at 15 polymorophic loci in Eurema butterflies Locus  Allele  E. mandarina  E. hecabe  E. blanda  Tokyo  Kochi  Fukuoka  Oki-Em  Oki-Eh  Ishigaki  Iriomote  EST-2  n  139  24  48  27  72  86  17  35    +  0  0  0.125  0  0.292  0.860  1.000  0.886    −  1.000  1.000  0.875  1.000  0.708  0.140  0  0.114  PGM  n  199  56  75  105  120  83  16  35    a  0.010  0  0  0  0  0.139  0.250  0    b  0.146  0.080  0.020  0.086  0.029  0.102  0.250  0    c  0.515  0.321  0.660  0.357  0.404  0.452  0.219  0.400    d  0.284  0.277  0.320  0.443  0.512  0.217  0.250  0.600    e  0.040  0.125  0  0.105  0.050  0.066  0  0    f  0.005  0.018  0  0.010  0.004  0  0  0    g  0  0  0  0  0  0.024  0.031  0    h  0.631  0.798  0.462  0.658  0.571  0.714  0.764  0.48  G6PD  n  183  51  107  120  118  81  17  35    a  0  0  0  0.079  0.640  0.512  0.618  0    b  0.014  0  0  0.071  0.161  0.327  0.235  1.000    c  0.975  0.990  1.000  0.842  0.178  0.130  0.147  0    d  0.005  0.010  0  0.008  0  0.019  0  0    e  0.005  0  0  0  0.021  0.012  0  0    h  0.048  0.019  0  0.28  0.533  0.613  0.542  0  LAP-1  n  68  28  35  24  73  30  17  34    a  0.191  0.375  0.071  0.146  0.192  0.100  0.029  0    b  0.390  0.143  0.643  0.479  0.521  0.617  0.500  0    c  0.324  0.482  0.129  0.354  0.178  0.217  0.382  0    d  0.074  0  0.157  0.021  0.110  0.067  0.088  0    e  0.007  0  0  0  0  0  0  0    f  0.015  0  0  0  0  0  0  0.853    g  0  0  0  0  0  0  0  0.147    h  0.701  0.607  0.54  0.623  0.649  0.558  0.595  0.251  LAP-2  n  74  26  35  22  75  21  15  33    a  0.966  0.942  0.957  0.977  0.840  0.643  0.833  0    b  0.034  0.058  0.043  0.023  0.153  0.214  0.133  0    c  0  0  0  0  0.007  0.119  0  0.197    d  0  0  0  0  0  0.024  0.033  0.682    e  0  0  0  0  0  0  0  0.121    h  0.065  0.109  0.082  0.044  0.271  0.526  0.287  0.482  aGPD1  n  122  119  86  54  91  68  17  35    a  0.885  0.874  0.983  0.944  0.967  0.941  1.000  0    b  0.102  0.105  0.017  0.046  0.033  0.059  0  0    c  0.004  0.004  0  0  0  0  0  0    d  0.008  0  0  0  0  0  0  0.829    e  0  0  0  0  0  0  0  0.171    f  0  0.017  0  0.009  0  0  0  0    h  0.206  0.225  0.034  0.106  0.064  0.111  0  0.284  aGPD2  n  122  120  86  54  93  67  17  35    a  0.828  0.829  0.936  0.870  0.871  0.843  1.000  0.986    b  0.156  0.15  0.064  0.130  0.129  0.149  0  0    c  0.008  0.008  0  0  0  0  0  0    d  0.008  0  0  0  0  0.007  0  0.014    e  0  0  0  0  0  0  0  0    f  0  0.013  0  0  0  0  0  0    h  0.29  0.29  0.12  0.226  0.225  0.267  0  0.028  HK-1  n  32  18  30  30  30  30  17  35    a  1.000  1.000  1.000  1.000  1.000  1.000  1.000  0    b  0  0  0  0  0  0  0  1.000    h  0  0  0  0  0  0  0  0  HK-2  n  32  18  30  30  30  30  17  35    a  1.000  1.000  1.000  1.000  1.000  1.000  1.000  0    b  0  0  0  0  0  0  0  1.000    h  0  0  0  0  0  0  0  0  GOT  n  163  41  10  63  82  69  17  17    a  0.009  0.037  0.100  0.381  0.067  0.014  0  0    b  0.957  0.902  0.900  0.619  0.933  0.551  0.118  0    c  0.034  0.024  0  0  0  0.428  0.882  0    d  0  0  0  0  0  0.007  0  0    e  0  0.037  0  0  0  0  0  1.000    h  0.083  0.182  0.180  0.472  0.125  0.514  0.208  0  ME  n  156  53  50  63  74  62  15  34    a  0.003  0  0  0  0.101  0.210  0.133  0.515    b  0.061  0.047  0  0.095  0.405  0.492  0.233  0.044    c  0.321  0.538  0.010  0.603  0.432  0.218  0.40  0.044    d  0.519  0.377  0.860  0.294  0.047  0.065  0.233  0    e  0.096  0.019  0.130  0.008  0.014  0.016  0  0    f  0  0.019  0  0  0  0  0  0.397    h  0.615  0.566  0.243  0.541  0.636  0.662  0.713  0.574  ICDH-1  n  44  59  20  40  18  21  15  35    a  0.864  0.788  0.750  0.700  0  0.143  0.200  0    b  0.125  0.186  0.250  0.213  0.944  0.833  0.800  0.786    c  0  0.017  0  0.025  0  0  0  0.214    d  0.011  0.008  0  0.038  0.056  0.024  0  0    e  0  0  0  0.013  0  0  0  0    f  0  0  0  0.013  0  0  0  0    h  0.238  0.344  0.375  0.463  0.105  0.285  0.32  0.337  ICDH-2  n  43  58  20  40  24  34  17  35    a  0.326  0  1.000  0.087  0.854  0.132  0.029  0.143    b  0.012  0  0  0.013  0.125  0.765  0.794  0.857    c  0.012  0  0  0  0.021  0.015  0  0    d  0.651  1.000  0  0.900  0  0.088  0.176  0    h  0.470  0  0  0.182  0.254  0.39  0.337  0.245  MDH  n  28  22  28  28  28  28  17  35    a  1.000  1.000  1.000  1.000  1.000  1.000  1.000  0    b  0  0  0  0  0  0  0  1.000    h  0  0  0  0  0  0  0  0  6PGD  n  86  30  23  44  80  38  17  31    a  0  0  0  0.034  0.913  0.776  0.647  0.065    b  0.052  0.050  0  0.114  0.025  0.224  0.324  0.935    c  0.913  0.917  1.000  0.852  0.063  0  0.029  0    d  0.035  0.033  0  0  0  0  0  0    h  0.163  0.156  0  0.260  0.163  0.347  0.476  0.121  Locus  Allele  E. mandarina  E. hecabe  E. blanda  Tokyo  Kochi  Fukuoka  Oki-Em  Oki-Eh  Ishigaki  Iriomote  EST-2  n  139  24  48  27  72  86  17  35    +  0  0  0.125  0  0.292  0.860  1.000  0.886    −  1.000  1.000  0.875  1.000  0.708  0.140  0  0.114  PGM  n  199  56  75  105  120  83  16  35    a  0.010  0  0  0  0  0.139  0.250  0    b  0.146  0.080  0.020  0.086  0.029  0.102  0.250  0    c  0.515  0.321  0.660  0.357  0.404  0.452  0.219  0.400    d  0.284  0.277  0.320  0.443  0.512  0.217  0.250  0.600    e  0.040  0.125  0  0.105  0.050  0.066  0  0    f  0.005  0.018  0  0.010  0.004  0  0  0    g  0  0  0  0  0  0.024  0.031  0    h  0.631  0.798  0.462  0.658  0.571  0.714  0.764  0.48  G6PD  n  183  51  107  120  118  81  17  35    a  0  0  0  0.079  0.640  0.512  0.618  0    b  0.014  0  0  0.071  0.161  0.327  0.235  1.000    c  0.975  0.990  1.000  0.842  0.178  0.130  0.147  0    d  0.005  0.010  0  0.008  0  0.019  0  0    e  0.005  0  0  0  0.021  0.012  0  0    h  0.048  0.019  0  0.28  0.533  0.613  0.542  0  LAP-1  n  68  28  35  24  73  30  17  34    a  0.191  0.375  0.071  0.146  0.192  0.100  0.029  0    b  0.390  0.143  0.643  0.479  0.521  0.617  0.500  0    c  0.324  0.482  0.129  0.354  0.178  0.217  0.382  0    d  0.074  0  0.157  0.021  0.110  0.067  0.088  0    e  0.007  0  0  0  0  0  0  0    f  0.015  0  0  0  0  0  0  0.853    g  0  0  0  0  0  0  0  0.147    h  0.701  0.607  0.54  0.623  0.649  0.558  0.595  0.251  LAP-2  n  74  26  35  22  75  21  15  33    a  0.966  0.942  0.957  0.977  0.840  0.643  0.833  0    b  0.034  0.058  0.043  0.023  0.153  0.214  0.133  0    c  0  0  0  0  0.007  0.119  0  0.197    d  0  0  0  0  0  0.024  0.033  0.682    e  0  0  0  0  0  0  0  0.121    h  0.065  0.109  0.082  0.044  0.271  0.526  0.287  0.482  aGPD1  n  122  119  86  54  91  68  17  35    a  0.885  0.874  0.983  0.944  0.967  0.941  1.000  0    b  0.102  0.105  0.017  0.046  0.033  0.059  0  0    c  0.004  0.004  0  0  0  0  0  0    d  0.008  0  0  0  0  0  0  0.829    e  0  0  0  0  0  0  0  0.171    f  0  0.017  0  0.009  0  0  0  0    h  0.206  0.225  0.034  0.106  0.064  0.111  0  0.284  aGPD2  n  122  120  86  54  93  67  17  35    a  0.828  0.829  0.936  0.870  0.871  0.843  1.000  0.986    b  0.156  0.15  0.064  0.130  0.129  0.149  0  0    c  0.008  0.008  0  0  0  0  0  0    d  0.008  0  0  0  0  0.007  0  0.014    e  0  0  0  0  0  0  0  0    f  0  0.013  0  0  0  0  0  0    h  0.29  0.29  0.12  0.226  0.225  0.267  0  0.028  HK-1  n  32  18  30  30  30  30  17  35    a  1.000  1.000  1.000  1.000  1.000  1.000  1.000  0    b  0  0  0  0  0  0  0  1.000    h  0  0  0  0  0  0  0  0  HK-2  n  32  18  30  30  30  30  17  35    a  1.000  1.000  1.000  1.000  1.000  1.000  1.000  0    b  0  0  0  0  0  0  0  1.000    h  0  0  0  0  0  0  0  0  GOT  n  163  41  10  63  82  69  17  17    a  0.009  0.037  0.100  0.381  0.067  0.014  0  0    b  0.957  0.902  0.900  0.619  0.933  0.551  0.118  0    c  0.034  0.024  0  0  0  0.428  0.882  0    d  0  0  0  0  0  0.007  0  0    e  0  0.037  0  0  0  0  0  1.000    h  0.083  0.182  0.180  0.472  0.125  0.514  0.208  0  ME  n  156  53  50  63  74  62  15  34    a  0.003  0  0  0  0.101  0.210  0.133  0.515    b  0.061  0.047  0  0.095  0.405  0.492  0.233  0.044    c  0.321  0.538  0.010  0.603  0.432  0.218  0.40  0.044    d  0.519  0.377  0.860  0.294  0.047  0.065  0.233  0    e  0.096  0.019  0.130  0.008  0.014  0.016  0  0    f  0  0.019  0  0  0  0  0  0.397    h  0.615  0.566  0.243  0.541  0.636  0.662  0.713  0.574  ICDH-1  n  44  59  20  40  18  21  15  35    a  0.864  0.788  0.750  0.700  0  0.143  0.200  0    b  0.125  0.186  0.250  0.213  0.944  0.833  0.800  0.786    c  0  0.017  0  0.025  0  0  0  0.214    d  0.011  0.008  0  0.038  0.056  0.024  0  0    e  0  0  0  0.013  0  0  0  0    f  0  0  0  0.013  0  0  0  0    h  0.238  0.344  0.375  0.463  0.105  0.285  0.32  0.337  ICDH-2  n  43  58  20  40  24  34  17  35    a  0.326  0  1.000  0.087  0.854  0.132  0.029  0.143    b  0.012  0  0  0.013  0.125  0.765  0.794  0.857    c  0.012  0  0  0  0.021  0.015  0  0    d  0.651  1.000  0  0.900  0  0.088  0.176  0    h  0.470  0  0  0.182  0.254  0.39  0.337  0.245  MDH  n  28  22  28  28  28  28  17  35    a  1.000  1.000  1.000  1.000  1.000  1.000  1.000  0    b  0  0  0  0  0  0  0  1.000    h  0  0  0  0  0  0  0  0  6PGD  n  86  30  23  44  80  38  17  31    a  0  0  0  0.034  0.913  0.776  0.647  0.065    b  0.052  0.050  0  0.114  0.025  0.224  0.324  0.935    c  0.913  0.917  1.000  0.852  0.063  0  0.029  0    d  0.035  0.033  0  0  0  0  0  0    h  0.163  0.156  0  0.260  0.163  0.347  0.476  0.121  View Large Results The zymograms of electrophoresis obtained were different among the two species of butterflies. Figure 1 shows zymograms of esterases on the both Eurema species. The zymograms of esterases were divided into two estimated loci, which were called Est-1 and Est-2, respectively. The Est-2 bands appeared in the Ishigaki-jima population of E. hecabe but not in the Tokyo population of E. mandarina. We examined the appearance of Est-2 bands on the gel among populations of two species used in this study. Figure 2 shows the percentage of appearance of Est-2 in both species. The Est-2 bands appeared in almost all individuals in Ishigaki-jima and Iriomote-jima Islands. The Est-2 bands also appeared on the gel in 88.6 to 100% of individuals in Ryukyu Islands. In the Tokyo population, however, the Est-2 bands appeared in none of the individuals, and in the Fukuoka population, it appeared in about 10% of individuals. Fig. 1. View largeDownload slide Esterases zymograms of E. mandarina (Left) and E. hecabe. Fig. 1. View largeDownload slide Esterases zymograms of E. mandarina (Left) and E. hecabe. Fig. 2. View largeDownload slide Percent appearance of Est-2 in Eurema butterflies Em: Eurema mandarina, Eh: E. hecabe. Fig. 2. View largeDownload slide Percent appearance of Est-2 in Eurema butterflies Em: Eurema mandarina, Eh: E. hecabe. Figure 3 shows the zymogram of G6PD in six populations of E. mandarina and E. hecabe. From the phenotype of the band (allele), we estimated that these bands were controlled by the dimeric enzyme in a single locus. Five alleles (a, b, c, d and e) were detected on G6PD locus. Table 2 shows allele frequencies of 15 loci from 11 enzymes of five populations of E. mandarina and three populations of E. hecabe. In the case of G6PD locus in Table 2, the allele ‘d’ in populations of E. mandarina appeared most frequently. On the other hand, alleles ‘a’ and ‘b’ frequently appeared on populations of E. hecabe. A similar tendency on allele frequencies was observed also in E. mandarina and E. hecabe sympatrically distributed in Okinawa Island. Fig. 3. View largeDownload slide G6PD zymogram of E. mandarina (Left) and E. hecabe. Fig. 3. View largeDownload slide G6PD zymogram of E. mandarina (Left) and E. hecabe. We obtained UPGMA and NJ phylogenitic trees from allele frequencies using the POPTREE2 software (Takezaki et al. 2010). All the UPGMA trees showed the same topology—the Eurema butterflies used in this study were divided into two groups: E. hecabe and E. mandarina. Also the NJ trees using Fst distance (Latter 1972) showed the same topology as the UPGMA trees. However, when using GST distance data (Nei 1973), the populations of E. mandarina formed one clade, whereas the population of E. hecabe showed paraphyletical relationship. Figure 4 shows NJ tree using FST distance. The four populations of E. mandarina formed one clade supported by high bootstrap values, and also three populations of E. hecabe created one clade not supported by high bootstrap values. Fig. 4. View largeDownload slide Neighbor-Joining tree of E. mandarina and E. hecabe using FST distance. Numbers are bootstrap values obtained from 1,000 replicates. Fig. 4. View largeDownload slide Neighbor-Joining tree of E. mandarina and E. hecabe using FST distance. Numbers are bootstrap values obtained from 1,000 replicates. Discussion Simple identification methods using an allozyme zymogram as shown in our study have been carried out in the past (e.g., Nomura and Ichinose 1990, Goka and Takafuji 1997). This study also showed that it is possible to distinguish between two closely related butterfly species—E. hecabe and E. mandarina at several loci. When comparing the two species used in this study, the Est-2 band appeared in most individuals of E. mandarina, but it was rarely seen in E. hecabe. The phylogenetic trees obtained clearly show that populations of Japanese E. hecabe and E. mandarina are classified into two genetically different groups, which are linked by several characters, such as seasonal expression of wing morph, mate recognition and host-plant use (Kato 2000a,b; Kabayashi et al. 2001). Phylogenetic analysis using allele frequencies has shown many useful results so far (e.g., Ayala and Powell 1972, Roininen et al. 1993, Nomura 1998). In the Japanese fauna of E. hecabe and E. mandarina, it was clarified that the mitochondria of E. hecabe has introgressed to E. mandarina by the selective sweep of the Wolbachia infection (Narita et al. 2006). For this reason, most of the mtDNA of E. hecabe is the same as in E. mandarina, so that there is no clear distinction between the two species. Also the DNA barcoding method (Hebert et al. 2003) utilizing the gene sequence of mtDNA can not correctly identify either species (Narita et al. 2006). In this study, the results of analyzing allele frequency in allozyme showed that there can be clear distinction between the two closely related species of Eurema butterflies, which could not be identified by mtDNA sequencies. All of the enzymes used in this study were encoded by nuclear genes, so it was confirmed that they are not affected by Wolbachia infection. Also both species sympatrically distributed on Okinawa Island could be divided appropriately. Currently, DNA analysis is a common laboratory method and analysis using nuclear DNA is also becoming common method, but as described in this research, it is also known that closely related species can not be identified using mtDNA because of selective sweep with symbiotic microorganisms (e.g., Jiggins 2003, Gompert et al. 2008, Graham and Wilson 2012), and that the species can not be correctly identified even by using the DNA barcoding method established as an useful identification method. In such species, nuclear genes will be used for molecular analysis, and it will be possible to use allozyme analysis for simplicity. Regarding molecular phylogenetic analysis, the usefulness of allozymes has been demonstrated in several insects so far now that DNA analysis is more common and can obtain more genetic information. However, in cases where analysis with organelle genes is difficult due to selective sweep, traditional methods may be useful as shown in this study. We used a traditional method of allozyme variation analysis in this study, which was also effective in identifying closely related species that had been confused using methods involving mtDNA sequences, such as DNA barcoding. Although the traditional method is less frequently used now, it can be utilized by selecting and using polymorphic enzymes. In addition, since it is possible to identify both species by examining the appearance of Est-2 (Fig. 1), a simple identification method of allozymes can be developed for the ecological investigation of the Southwest Islands in Japan where both butterflies are sympatrically distributed. And there are some methods—microsatellites (Harper et al. 2006, Habel et al. 2009, Saarinen and Daniels 2012), or RADSeq (Dupuis and Sperling 2015)—that might be employed to closely species that may be suitable for use with old specimens. Then, we could PCR- amplify DNA region such as G6PD that we showed difference in Eurema butterflies (Fig. 3) and then either sequence the amplified products, or distinguish between alleles by a restriction-enzyme based assay followed by electrophoresis (Gemmell and Marcus 2015). 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Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of the Entomological Society of America Oxford University Press

Allozyme Analyses of Two Closely Related Species of Eurema Butterflies (Lepidoptera: Pieridae) That Cannot Be Identified With Mitochondrial DNA Sequences

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Entomological Society of America
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© The Author(s) 2018. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
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10.1093/aesa/say001
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Abstract

Abstract The Japanese fauna of the yellow butterfly Eurema hecabe (L.) (Lepidoptera; Pieridae) populations in Ryukyu area (Southwestern Islands) were shown to differ from their Mainlands population (Honshu) in its seasonal forms of adult wing color pattern expression and in larval food habits. Recently, E. hecabe was divided into two species, E. hecabe and Eurema mandarina (de l’Orza) (Lepidoptera: Pieridae), based on their morphological, behavioral and genetic characteristics. However, whereas these two species are classified into two distinct groups based on nuclear DNA, both species are mixed in the phylogenetic tree from their mitochondrial DNA (mtDNA) sequences. The reason is a selective sweep by Wolbachia infection, and they can not be distinguished by DNA barcode method using mtDNA. We analyzed allozyme variation as a nuclear gene phenotype in 15 loci from 11 enzymes of the two closely related butterflies. Examining the esterase zymogram of each population, appearance frequency of Est-2 locus in each population varied greatly. E. mandarina showed the bands but E. hecabe showed no bands in Est-2 locus. The UPGMA and NJ phylogenetic trees branched out into two species—E. hecabe and E. mandarina—although their habitat overlapped in Okinawajima Is. populations. The results showed that the allozyme analyses were not affected by the selective sweep of Wolbachia infection between these two species. Geographic distribution of a natural population has historical and ecological components (Futuyma 1998). Genetic and ecological studies will provide available data for understanding how a single population in the past may have evolved into different populations within the same species or belong to closely related species widely distributed through different climatic zones. Eurema hecabe (L.) (Lepidoptera; Pieridae) is widely distributed from the tropical through subtropical to temperate regions in the Old world (Yata 1989). This species is a multivoltine butterfly and shows seasonal polyphenism (Summer/Wet morph and Autumn/Dry morph) in wing color pattern, which is associated with the reproductive diapause. Yata (1989) reported that populations of E. hecabe distributed in East Asia, including the Japanese population, belong to a single group on the basis of morphological characteristics, although the wing color pattern varies clinally with latitude. However, in Japan, which is the northern limit of the distribution area of E. hecabe, this species consists of two geographically separated types differing in fringe color (brown or yellow) of the upperside forewing (Kato 1999). These types are easily discriminated in the wing pattern of the autumn morph, although that of the summer morph is similar in appearance (Kato and Sano 1987, Kato and Handa 1992). Use of the host plants by larvae was also shown to differ between the two types (Kato et al. 1992). The brown type inhabits the subtropics of the Ryukyu Islands, and the yellow type inhabits mainly the temperate region including the Mainlands (e.g., Honshu) of Japan. Kato (1999) found that the butterflies of these two types sympatrically inhabit the subtropical Okinawa Island, which is in the northern part of the Ryukyu Islands. Their distribution on this island basically depends on the locality of their host-plant species. Laboratory experiments showed that females of each type produced offspring with the same fringe color and seasonal wing morph as their parents, and the ability for host utilization differed between them on this island. Therefore, it seems likely that there are two distinct populations of E. hecabe. Because of differences mentioned above, the two types of E. hecabe were judged to be of two different species, and the yellow type was named Eurema mandarina (de l’Orza) (Lepidoptera: Pieridae) (Shirouzu 2006). In the molecular phylogenetic analysis, these two species can be divided into two groups in the nucleotide sequence of nuclear DNA, but in the case of mitochondrial DNA (mtDNA), the two species were mixed in the phylogenetic tree and could not be clearly divided into two groups (Narita et al. 2006). It was revealed that mismatch of systematic relationship was caused by selective sweep by Wolbachia infection (Narita et al. 2006). For this reason, identifying these two species using mtDNA sequence is impossible because they have almost the same mtDNA sequence, and the two species can not be distinguished when using the DNA barcoding region (Hebert et al. 2003). Also, since nuclear DNA has heterozygous sequences from male and female chromosome, sometimes it requires an extra process: cloning. In addition, identification of the two Eurema butterflies in sympatric distribution areas is quite difficult using old specimens. Therefore, we attempted to classify the two groups by gene frequency analysis of allozymes, which is a traditional molecular phylogenetic method. To study the genetic variation and phylogeny of biological organisms including insects, a biochemical or molecular approach has been accepted as useful tools. Analysis of allozyme variation is also one of them used for clarifying genetic differences among populations within a species and among closely related species (e.g., Avise 1975, 1993; Ayala 1975; Jacobson et al. 1981; Pashley 1983; Nomura 1998). In the present study, we analyzed allozyme variation for the sympatric and allopatric populations of this species, by means of the polycarylamide gel electrophoretic technique for clarifying the genetic relationships among populations of E. hecabe and E. mandarina in Japan. Materials and Methods Insects Adult males and females of E. hecabe and E. mandarina were caught from 1991 to 1996 at different locations (Mainlands and Ryukyu [Southwest] Islands) in Japan (Table 1), and the identified species were recorded primarily by the fringe color (yellow or brown) of the upperside forewing. As an outgroup species for phylogenetic analyses, Eurema blanda (Boisduval) (Lepidoptera: Pieridae) was used, which were caught in Ishigaki Is. Then, the individuals that were frozen and stored at -80°C were used for analysis. We used the old samples for the experiment, because it is almost impossible to collect E. mandarina population in Rykyu Islands as a result of recent urban development projects. Table 1. Populations used and localities of collection in this study Species population  Locality  Latitude: longitude  Date  E. mandarina   Tokyo  Mitaka  N 35°41′: E 139°46′  Aug., 1991–1993   Shikoku  Kochi  N 33°32′: E 133°28′  July–Nov., 1993   Kyushu  Fukuoka  N 33°37′: E 130°25′  Sept., 1992   Oki-Em  Okinawa-jima  N 26°17′: E 127°47′  Oct., 1992, 1993  E. hecabe   Oki-Eh  Okinawa-jima  N 26°17′: E 127°47′  Mar.–Oct., 1991–1994   Ishigaki  Ishigaki-jima  N 24°20′: E 124°09′  Mar.–Oct., 1991–1992   Iriomote  Iriomote-jima  N 24°17′: E 123°51′  May, 1996  E. blanda (outgroup)   Ishigaki  Ishigaki-jima  N 24°20′: E 124°09′  May, 1996  Species population  Locality  Latitude: longitude  Date  E. mandarina   Tokyo  Mitaka  N 35°41′: E 139°46′  Aug., 1991–1993   Shikoku  Kochi  N 33°32′: E 133°28′  July–Nov., 1993   Kyushu  Fukuoka  N 33°37′: E 130°25′  Sept., 1992   Oki-Em  Okinawa-jima  N 26°17′: E 127°47′  Oct., 1992, 1993  E. hecabe   Oki-Eh  Okinawa-jima  N 26°17′: E 127°47′  Mar.–Oct., 1991–1994   Ishigaki  Ishigaki-jima  N 24°20′: E 124°09′  Mar.–Oct., 1991–1992   Iriomote  Iriomote-jima  N 24°17′: E 123°51′  May, 1996  E. blanda (outgroup)   Ishigaki  Ishigaki-jima  N 24°20′: E 124°09′  May, 1996  View Large An individual thorax (without wings and legs) was excised, and homogenized with 0.3 ml of 0.01 M phosphate buffer (pH 7.0) in a 1.5 ml disposable microcentrifuge tube and pellet pestle. The homogenates were centrifuged at 10,000×g for 20 min. under 4°C. The supernatants were stored at −40°C until electrophoresis. Electrophoresis and Analyses Electrophoresis was performed on a 7.5% polyacrylamide gel by the method of Davis (1964) using a vertical slab gel apparatus (Model AE6200: ATTO Corp., Tokyo) as described previously (Nomura and Ichinose 1990). After electrophoresis, the gels were stained for 11 enzymes as follows: esterases (EST, EC 3.1.1.-), glucose-6-phoshate dehydrogenase (G6PD, EC 1.1.1.49), glutamate oxaloacetate transaminase (GOT, EC 2.6.1.1), α-glycerophosphate dehydrogenase (α-GPDH, EC 1.1.1.8), hexokinase (HK, EC 2.7.1.1), isocitrate dehydrogenase (ICDH, EC 1.1.1.42), leucine amino peptidase (LAP, EC 3.4.11.1), malic dehydrogenase (MDH, EC 1.1.1.37), malic enzyme (ME, EC 1.1.1.40), phosphoglucomutase (PGM, EC 2.7.5.1) and 6-phoshogluconate dehydrogenase (6PGDH, EC 1.1.1.44). The staining methods used for all enzymes were those of Steiner and Joslyn (1979) with some modifications, except the staining methods for ME and EST, which were those of Loxdale et al. (1983) and Nomura and Ichinose (1990), respectively, with some modifications. We obtained zymograms of electrophoretic gels of 11 enzymes mentioned above, and estimated bands that were controlled by single locus. From our preliminary study, the appearance of bands of 11 enzymes on the gel did not differ between sexes and among ages of butterflies. We obtained 15 loci from the 11 enzymes (Table 2) and calculated the allele frequencies of 15 loci from electrophoretic data, and analyzed with POPTREE2 (Takezaki et al. 2010) to obtain Genetic Distance measures and the UPGMA (Unweighted pair strain method of arithmetic average; Sneath and Sokal 1973) and NJ (Neighbor-Joining) phylogenetic trees (Saitou and Nei 1987). Table 2. Allozyme frequencies at 15 polymorophic loci in Eurema butterflies Locus  Allele  E. mandarina  E. hecabe  E. blanda  Tokyo  Kochi  Fukuoka  Oki-Em  Oki-Eh  Ishigaki  Iriomote  EST-2  n  139  24  48  27  72  86  17  35    +  0  0  0.125  0  0.292  0.860  1.000  0.886    −  1.000  1.000  0.875  1.000  0.708  0.140  0  0.114  PGM  n  199  56  75  105  120  83  16  35    a  0.010  0  0  0  0  0.139  0.250  0    b  0.146  0.080  0.020  0.086  0.029  0.102  0.250  0    c  0.515  0.321  0.660  0.357  0.404  0.452  0.219  0.400    d  0.284  0.277  0.320  0.443  0.512  0.217  0.250  0.600    e  0.040  0.125  0  0.105  0.050  0.066  0  0    f  0.005  0.018  0  0.010  0.004  0  0  0    g  0  0  0  0  0  0.024  0.031  0    h  0.631  0.798  0.462  0.658  0.571  0.714  0.764  0.48  G6PD  n  183  51  107  120  118  81  17  35    a  0  0  0  0.079  0.640  0.512  0.618  0    b  0.014  0  0  0.071  0.161  0.327  0.235  1.000    c  0.975  0.990  1.000  0.842  0.178  0.130  0.147  0    d  0.005  0.010  0  0.008  0  0.019  0  0    e  0.005  0  0  0  0.021  0.012  0  0    h  0.048  0.019  0  0.28  0.533  0.613  0.542  0  LAP-1  n  68  28  35  24  73  30  17  34    a  0.191  0.375  0.071  0.146  0.192  0.100  0.029  0    b  0.390  0.143  0.643  0.479  0.521  0.617  0.500  0    c  0.324  0.482  0.129  0.354  0.178  0.217  0.382  0    d  0.074  0  0.157  0.021  0.110  0.067  0.088  0    e  0.007  0  0  0  0  0  0  0    f  0.015  0  0  0  0  0  0  0.853    g  0  0  0  0  0  0  0  0.147    h  0.701  0.607  0.54  0.623  0.649  0.558  0.595  0.251  LAP-2  n  74  26  35  22  75  21  15  33    a  0.966  0.942  0.957  0.977  0.840  0.643  0.833  0    b  0.034  0.058  0.043  0.023  0.153  0.214  0.133  0    c  0  0  0  0  0.007  0.119  0  0.197    d  0  0  0  0  0  0.024  0.033  0.682    e  0  0  0  0  0  0  0  0.121    h  0.065  0.109  0.082  0.044  0.271  0.526  0.287  0.482  aGPD1  n  122  119  86  54  91  68  17  35    a  0.885  0.874  0.983  0.944  0.967  0.941  1.000  0    b  0.102  0.105  0.017  0.046  0.033  0.059  0  0    c  0.004  0.004  0  0  0  0  0  0    d  0.008  0  0  0  0  0  0  0.829    e  0  0  0  0  0  0  0  0.171    f  0  0.017  0  0.009  0  0  0  0    h  0.206  0.225  0.034  0.106  0.064  0.111  0  0.284  aGPD2  n  122  120  86  54  93  67  17  35    a  0.828  0.829  0.936  0.870  0.871  0.843  1.000  0.986    b  0.156  0.15  0.064  0.130  0.129  0.149  0  0    c  0.008  0.008  0  0  0  0  0  0    d  0.008  0  0  0  0  0.007  0  0.014    e  0  0  0  0  0  0  0  0    f  0  0.013  0  0  0  0  0  0    h  0.29  0.29  0.12  0.226  0.225  0.267  0  0.028  HK-1  n  32  18  30  30  30  30  17  35    a  1.000  1.000  1.000  1.000  1.000  1.000  1.000  0    b  0  0  0  0  0  0  0  1.000    h  0  0  0  0  0  0  0  0  HK-2  n  32  18  30  30  30  30  17  35    a  1.000  1.000  1.000  1.000  1.000  1.000  1.000  0    b  0  0  0  0  0  0  0  1.000    h  0  0  0  0  0  0  0  0  GOT  n  163  41  10  63  82  69  17  17    a  0.009  0.037  0.100  0.381  0.067  0.014  0  0    b  0.957  0.902  0.900  0.619  0.933  0.551  0.118  0    c  0.034  0.024  0  0  0  0.428  0.882  0    d  0  0  0  0  0  0.007  0  0    e  0  0.037  0  0  0  0  0  1.000    h  0.083  0.182  0.180  0.472  0.125  0.514  0.208  0  ME  n  156  53  50  63  74  62  15  34    a  0.003  0  0  0  0.101  0.210  0.133  0.515    b  0.061  0.047  0  0.095  0.405  0.492  0.233  0.044    c  0.321  0.538  0.010  0.603  0.432  0.218  0.40  0.044    d  0.519  0.377  0.860  0.294  0.047  0.065  0.233  0    e  0.096  0.019  0.130  0.008  0.014  0.016  0  0    f  0  0.019  0  0  0  0  0  0.397    h  0.615  0.566  0.243  0.541  0.636  0.662  0.713  0.574  ICDH-1  n  44  59  20  40  18  21  15  35    a  0.864  0.788  0.750  0.700  0  0.143  0.200  0    b  0.125  0.186  0.250  0.213  0.944  0.833  0.800  0.786    c  0  0.017  0  0.025  0  0  0  0.214    d  0.011  0.008  0  0.038  0.056  0.024  0  0    e  0  0  0  0.013  0  0  0  0    f  0  0  0  0.013  0  0  0  0    h  0.238  0.344  0.375  0.463  0.105  0.285  0.32  0.337  ICDH-2  n  43  58  20  40  24  34  17  35    a  0.326  0  1.000  0.087  0.854  0.132  0.029  0.143    b  0.012  0  0  0.013  0.125  0.765  0.794  0.857    c  0.012  0  0  0  0.021  0.015  0  0    d  0.651  1.000  0  0.900  0  0.088  0.176  0    h  0.470  0  0  0.182  0.254  0.39  0.337  0.245  MDH  n  28  22  28  28  28  28  17  35    a  1.000  1.000  1.000  1.000  1.000  1.000  1.000  0    b  0  0  0  0  0  0  0  1.000    h  0  0  0  0  0  0  0  0  6PGD  n  86  30  23  44  80  38  17  31    a  0  0  0  0.034  0.913  0.776  0.647  0.065    b  0.052  0.050  0  0.114  0.025  0.224  0.324  0.935    c  0.913  0.917  1.000  0.852  0.063  0  0.029  0    d  0.035  0.033  0  0  0  0  0  0    h  0.163  0.156  0  0.260  0.163  0.347  0.476  0.121  Locus  Allele  E. mandarina  E. hecabe  E. blanda  Tokyo  Kochi  Fukuoka  Oki-Em  Oki-Eh  Ishigaki  Iriomote  EST-2  n  139  24  48  27  72  86  17  35    +  0  0  0.125  0  0.292  0.860  1.000  0.886    −  1.000  1.000  0.875  1.000  0.708  0.140  0  0.114  PGM  n  199  56  75  105  120  83  16  35    a  0.010  0  0  0  0  0.139  0.250  0    b  0.146  0.080  0.020  0.086  0.029  0.102  0.250  0    c  0.515  0.321  0.660  0.357  0.404  0.452  0.219  0.400    d  0.284  0.277  0.320  0.443  0.512  0.217  0.250  0.600    e  0.040  0.125  0  0.105  0.050  0.066  0  0    f  0.005  0.018  0  0.010  0.004  0  0  0    g  0  0  0  0  0  0.024  0.031  0    h  0.631  0.798  0.462  0.658  0.571  0.714  0.764  0.48  G6PD  n  183  51  107  120  118  81  17  35    a  0  0  0  0.079  0.640  0.512  0.618  0    b  0.014  0  0  0.071  0.161  0.327  0.235  1.000    c  0.975  0.990  1.000  0.842  0.178  0.130  0.147  0    d  0.005  0.010  0  0.008  0  0.019  0  0    e  0.005  0  0  0  0.021  0.012  0  0    h  0.048  0.019  0  0.28  0.533  0.613  0.542  0  LAP-1  n  68  28  35  24  73  30  17  34    a  0.191  0.375  0.071  0.146  0.192  0.100  0.029  0    b  0.390  0.143  0.643  0.479  0.521  0.617  0.500  0    c  0.324  0.482  0.129  0.354  0.178  0.217  0.382  0    d  0.074  0  0.157  0.021  0.110  0.067  0.088  0    e  0.007  0  0  0  0  0  0  0    f  0.015  0  0  0  0  0  0  0.853    g  0  0  0  0  0  0  0  0.147    h  0.701  0.607  0.54  0.623  0.649  0.558  0.595  0.251  LAP-2  n  74  26  35  22  75  21  15  33    a  0.966  0.942  0.957  0.977  0.840  0.643  0.833  0    b  0.034  0.058  0.043  0.023  0.153  0.214  0.133  0    c  0  0  0  0  0.007  0.119  0  0.197    d  0  0  0  0  0  0.024  0.033  0.682    e  0  0  0  0  0  0  0  0.121    h  0.065  0.109  0.082  0.044  0.271  0.526  0.287  0.482  aGPD1  n  122  119  86  54  91  68  17  35    a  0.885  0.874  0.983  0.944  0.967  0.941  1.000  0    b  0.102  0.105  0.017  0.046  0.033  0.059  0  0    c  0.004  0.004  0  0  0  0  0  0    d  0.008  0  0  0  0  0  0  0.829    e  0  0  0  0  0  0  0  0.171    f  0  0.017  0  0.009  0  0  0  0    h  0.206  0.225  0.034  0.106  0.064  0.111  0  0.284  aGPD2  n  122  120  86  54  93  67  17  35    a  0.828  0.829  0.936  0.870  0.871  0.843  1.000  0.986    b  0.156  0.15  0.064  0.130  0.129  0.149  0  0    c  0.008  0.008  0  0  0  0  0  0    d  0.008  0  0  0  0  0.007  0  0.014    e  0  0  0  0  0  0  0  0    f  0  0.013  0  0  0  0  0  0    h  0.29  0.29  0.12  0.226  0.225  0.267  0  0.028  HK-1  n  32  18  30  30  30  30  17  35    a  1.000  1.000  1.000  1.000  1.000  1.000  1.000  0    b  0  0  0  0  0  0  0  1.000    h  0  0  0  0  0  0  0  0  HK-2  n  32  18  30  30  30  30  17  35    a  1.000  1.000  1.000  1.000  1.000  1.000  1.000  0    b  0  0  0  0  0  0  0  1.000    h  0  0  0  0  0  0  0  0  GOT  n  163  41  10  63  82  69  17  17    a  0.009  0.037  0.100  0.381  0.067  0.014  0  0    b  0.957  0.902  0.900  0.619  0.933  0.551  0.118  0    c  0.034  0.024  0  0  0  0.428  0.882  0    d  0  0  0  0  0  0.007  0  0    e  0  0.037  0  0  0  0  0  1.000    h  0.083  0.182  0.180  0.472  0.125  0.514  0.208  0  ME  n  156  53  50  63  74  62  15  34    a  0.003  0  0  0  0.101  0.210  0.133  0.515    b  0.061  0.047  0  0.095  0.405  0.492  0.233  0.044    c  0.321  0.538  0.010  0.603  0.432  0.218  0.40  0.044    d  0.519  0.377  0.860  0.294  0.047  0.065  0.233  0    e  0.096  0.019  0.130  0.008  0.014  0.016  0  0    f  0  0.019  0  0  0  0  0  0.397    h  0.615  0.566  0.243  0.541  0.636  0.662  0.713  0.574  ICDH-1  n  44  59  20  40  18  21  15  35    a  0.864  0.788  0.750  0.700  0  0.143  0.200  0    b  0.125  0.186  0.250  0.213  0.944  0.833  0.800  0.786    c  0  0.017  0  0.025  0  0  0  0.214    d  0.011  0.008  0  0.038  0.056  0.024  0  0    e  0  0  0  0.013  0  0  0  0    f  0  0  0  0.013  0  0  0  0    h  0.238  0.344  0.375  0.463  0.105  0.285  0.32  0.337  ICDH-2  n  43  58  20  40  24  34  17  35    a  0.326  0  1.000  0.087  0.854  0.132  0.029  0.143    b  0.012  0  0  0.013  0.125  0.765  0.794  0.857    c  0.012  0  0  0  0.021  0.015  0  0    d  0.651  1.000  0  0.900  0  0.088  0.176  0    h  0.470  0  0  0.182  0.254  0.39  0.337  0.245  MDH  n  28  22  28  28  28  28  17  35    a  1.000  1.000  1.000  1.000  1.000  1.000  1.000  0    b  0  0  0  0  0  0  0  1.000    h  0  0  0  0  0  0  0  0  6PGD  n  86  30  23  44  80  38  17  31    a  0  0  0  0.034  0.913  0.776  0.647  0.065    b  0.052  0.050  0  0.114  0.025  0.224  0.324  0.935    c  0.913  0.917  1.000  0.852  0.063  0  0.029  0    d  0.035  0.033  0  0  0  0  0  0    h  0.163  0.156  0  0.260  0.163  0.347  0.476  0.121  View Large Results The zymograms of electrophoresis obtained were different among the two species of butterflies. Figure 1 shows zymograms of esterases on the both Eurema species. The zymograms of esterases were divided into two estimated loci, which were called Est-1 and Est-2, respectively. The Est-2 bands appeared in the Ishigaki-jima population of E. hecabe but not in the Tokyo population of E. mandarina. We examined the appearance of Est-2 bands on the gel among populations of two species used in this study. Figure 2 shows the percentage of appearance of Est-2 in both species. The Est-2 bands appeared in almost all individuals in Ishigaki-jima and Iriomote-jima Islands. The Est-2 bands also appeared on the gel in 88.6 to 100% of individuals in Ryukyu Islands. In the Tokyo population, however, the Est-2 bands appeared in none of the individuals, and in the Fukuoka population, it appeared in about 10% of individuals. Fig. 1. View largeDownload slide Esterases zymograms of E. mandarina (Left) and E. hecabe. Fig. 1. View largeDownload slide Esterases zymograms of E. mandarina (Left) and E. hecabe. Fig. 2. View largeDownload slide Percent appearance of Est-2 in Eurema butterflies Em: Eurema mandarina, Eh: E. hecabe. Fig. 2. View largeDownload slide Percent appearance of Est-2 in Eurema butterflies Em: Eurema mandarina, Eh: E. hecabe. Figure 3 shows the zymogram of G6PD in six populations of E. mandarina and E. hecabe. From the phenotype of the band (allele), we estimated that these bands were controlled by the dimeric enzyme in a single locus. Five alleles (a, b, c, d and e) were detected on G6PD locus. Table 2 shows allele frequencies of 15 loci from 11 enzymes of five populations of E. mandarina and three populations of E. hecabe. In the case of G6PD locus in Table 2, the allele ‘d’ in populations of E. mandarina appeared most frequently. On the other hand, alleles ‘a’ and ‘b’ frequently appeared on populations of E. hecabe. A similar tendency on allele frequencies was observed also in E. mandarina and E. hecabe sympatrically distributed in Okinawa Island. Fig. 3. View largeDownload slide G6PD zymogram of E. mandarina (Left) and E. hecabe. Fig. 3. View largeDownload slide G6PD zymogram of E. mandarina (Left) and E. hecabe. We obtained UPGMA and NJ phylogenitic trees from allele frequencies using the POPTREE2 software (Takezaki et al. 2010). All the UPGMA trees showed the same topology—the Eurema butterflies used in this study were divided into two groups: E. hecabe and E. mandarina. Also the NJ trees using Fst distance (Latter 1972) showed the same topology as the UPGMA trees. However, when using GST distance data (Nei 1973), the populations of E. mandarina formed one clade, whereas the population of E. hecabe showed paraphyletical relationship. Figure 4 shows NJ tree using FST distance. The four populations of E. mandarina formed one clade supported by high bootstrap values, and also three populations of E. hecabe created one clade not supported by high bootstrap values. Fig. 4. View largeDownload slide Neighbor-Joining tree of E. mandarina and E. hecabe using FST distance. Numbers are bootstrap values obtained from 1,000 replicates. Fig. 4. View largeDownload slide Neighbor-Joining tree of E. mandarina and E. hecabe using FST distance. Numbers are bootstrap values obtained from 1,000 replicates. Discussion Simple identification methods using an allozyme zymogram as shown in our study have been carried out in the past (e.g., Nomura and Ichinose 1990, Goka and Takafuji 1997). This study also showed that it is possible to distinguish between two closely related butterfly species—E. hecabe and E. mandarina at several loci. When comparing the two species used in this study, the Est-2 band appeared in most individuals of E. mandarina, but it was rarely seen in E. hecabe. The phylogenetic trees obtained clearly show that populations of Japanese E. hecabe and E. mandarina are classified into two genetically different groups, which are linked by several characters, such as seasonal expression of wing morph, mate recognition and host-plant use (Kato 2000a,b; Kabayashi et al. 2001). Phylogenetic analysis using allele frequencies has shown many useful results so far (e.g., Ayala and Powell 1972, Roininen et al. 1993, Nomura 1998). In the Japanese fauna of E. hecabe and E. mandarina, it was clarified that the mitochondria of E. hecabe has introgressed to E. mandarina by the selective sweep of the Wolbachia infection (Narita et al. 2006). For this reason, most of the mtDNA of E. hecabe is the same as in E. mandarina, so that there is no clear distinction between the two species. Also the DNA barcoding method (Hebert et al. 2003) utilizing the gene sequence of mtDNA can not correctly identify either species (Narita et al. 2006). In this study, the results of analyzing allele frequency in allozyme showed that there can be clear distinction between the two closely related species of Eurema butterflies, which could not be identified by mtDNA sequencies. All of the enzymes used in this study were encoded by nuclear genes, so it was confirmed that they are not affected by Wolbachia infection. Also both species sympatrically distributed on Okinawa Island could be divided appropriately. Currently, DNA analysis is a common laboratory method and analysis using nuclear DNA is also becoming common method, but as described in this research, it is also known that closely related species can not be identified using mtDNA because of selective sweep with symbiotic microorganisms (e.g., Jiggins 2003, Gompert et al. 2008, Graham and Wilson 2012), and that the species can not be correctly identified even by using the DNA barcoding method established as an useful identification method. In such species, nuclear genes will be used for molecular analysis, and it will be possible to use allozyme analysis for simplicity. Regarding molecular phylogenetic analysis, the usefulness of allozymes has been demonstrated in several insects so far now that DNA analysis is more common and can obtain more genetic information. However, in cases where analysis with organelle genes is difficult due to selective sweep, traditional methods may be useful as shown in this study. We used a traditional method of allozyme variation analysis in this study, which was also effective in identifying closely related species that had been confused using methods involving mtDNA sequences, such as DNA barcoding. Although the traditional method is less frequently used now, it can be utilized by selecting and using polymorphic enzymes. In addition, since it is possible to identify both species by examining the appearance of Est-2 (Fig. 1), a simple identification method of allozymes can be developed for the ecological investigation of the Southwest Islands in Japan where both butterflies are sympatrically distributed. And there are some methods—microsatellites (Harper et al. 2006, Habel et al. 2009, Saarinen and Daniels 2012), or RADSeq (Dupuis and Sperling 2015)—that might be employed to closely species that may be suitable for use with old specimens. Then, we could PCR- amplify DNA region such as G6PD that we showed difference in Eurema butterflies (Fig. 3) and then either sequence the amplified products, or distinguish between alleles by a restriction-enzyme based assay followed by electrophoresis (Gemmell and Marcus 2015). 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Annals of the Entomological Society of AmericaOxford University Press

Published: Mar 1, 2018

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