MATERIALS AND METHODS

Methods: This investigation was conducted in March-April, 2017. For the sampling method, a purposive sampling was used where the sampling technique is free roaming²⁹.

Fig. 1:	Research location (hotspot) O.c. toeantei on Morotai Island, North Maluku (Morotai Island in Numbers, 2016 and modified by researchers, 2019)

Fig. 2:	Species O.c. toeantei butterflies endemic to the Island of Morotai

Study area: The collection of O.c. toeantei is conducted in 3 different locations of altitude (140 masl, 360 masl, dan 600 masl) in Sopia village Morotai. The map distribution of O.c. toeantei is presented in the Fig. 1.

Specimen collection: Ornithoptera croesus toeantei butterfly specimens obtained were 24 individuals. The O.c. toeantei specimen is preserved by using kaffir (dry matter) to identify morphometric characters. The O.c. toeantei specimens were also analyzed molecular-RAPD in the Laboratory of Molecular and Cellular Biology in Brawijaya University as many as 3 male and 3 female individuals (Fig. 2).

Morphometric data measurement: The common standard measurement for the butterfly consist of the measurement of the head length, thoracic length, abdominal length, antenna length, wing length and wing span^7,16,24,30. In addition to the standard characters in the form of body size, measurements of venation of wings were also carried out.

Measurements on butterfly body size include:

The measurement of the wing venation, including:

All wing venation characters were measured from the tip of the wing venation to the base of the wing venation^7,16,24,30.

Molecular-RAPD data measurement: The DNA was isolated using the Intron miniprep DNA kit. The DNA amplification at pradenaturation condition at 920°C for 4 min, denaturation at 920°C for 2 min, annealing at 360°C for 1 min 30 sec, extension at 720°C for 2 min and post extension at 720°C for 10 min for 45 cycles by using the Takara PCR with OPA 1 primers to OPA 10 (Table 3). Molecular data is based on the presence or absence of DNA bands with provisions value of 0 for no band and 1 for the presence of DNA bands.

The data obtained from morphometric and molecular measurements were analyzed descriptively quantitatively by using UPGMA cluster analysis (Unweight Pair Group Method with Arithmetic Mean) and the Multivariate Statistical Package (MVSP) program 3.22)³¹.

RESULT

Morphometric data: The data of this study is the average data from the morphological characters measurement (quantitative) of Morotai Island endemic butterflies with a total of 42 morphological characters from 24 individuals of O.c. toeantei (12 male and 12 female individuals). Then analyzed in similarities between individuals which consisted of analysis of matrix similarities (genetic distance) and dendrogram analysis to explain the diversity of intraspecies O.c. toeantei. Data from this analysis for both male and female of O.c. toeantei butterflies are shown in Table 1.

In the altitude of 140 masl, O.c. toeantei has the highest matrix similarity (coefficient similarity), which is 0.725. This implies that there are most similarities in the morphological character. The lowest matrix similarity (coefficient of similarity) is 0.431 for O.c. toeantei ♂ at the altitude of 600 masl and O.c. toeantei ♂ at an altitude of 140 masl. This implies that based on its morphological characters, O.c. toeantei at different altitude has fewest similarities in morphological characters. Furthermore, based on the results of the matrix similarities as mentioned before a dendrogram can be arranged which shows the group of individuals in the clusters as presented in Fig. 3.

The results of UPGMA analysis (dendrogram) showed that at the similarity value of 0.56, 2 main clusters of O.c. toeantei at an altitudes of 140 masl, 360 masl and 600 masl were formed.

Fig. 3:	Dendrogram data of 24 individuals O. c. toeantei based on morphological characters analyzed using the UPGMA method through the multivariate statistical package (MVSP) program 32

Table 1:	Similarity data of morphological characters O.c. toeantei Morotai Island endemic butterfly based on morphological characters analyzed using UPGMA cluster
♂: Male, ♀: Female

Table 2:	RAPD primary data sequence and polymorphic (%) O.c. toeantei based on the presence of the DNA-RAPD band pattern analyzed by the UPGMA method

The main cluster I with a similarity value of 0.70 consisted of O.c. toeantei ♀ 600 masl and O.c. toeantei ♂ 600 masl. The main cluster II with a similarity value of 0.62 formed into 2 subclusters, namely subcluster I with a similarity value of 0.67 consisting of O.c. toeantei ♀ 360 masl and O.c. toeantei ♂ 360 masl and subcluster II with a similarity value of 0.72 consisting of O.c. toeantei ♀ 140 masl and O.c. toeantei ♂ 140 masl. Thus, it is known that the most morphological characters are found on O.c. toeantei at the altitude of 140 masl and the lowest similarity at the altitude of 360 masl.

PCR-RAPD molecular data: The data of Molecular-RAPD character is achieved from the calculation of DNA bands appearance of O.c. toeantei with the total sample of 6 individuals (3 male and 3 female individuals) at different altitudes (140 masl, 360 masl and 600 masl ) (Table 2).

The total bands resulting from this study are 60 bands with criteria of 42 of polymorphic and 18 of monomorphic. Those results were identified from the DNA's band pattern appeared from the DNA photo. The average percentage of polymorphic is 76, 246% on OPA 1-10 primer (Table 3). Next, a further analysis was conducted on the matrix similarity based on the appearance of DNA band (DNA profile) with 1 as the score for DNA band appearance and 0 if the DNA band does not appears in every OPA 1-10 primer (Fig. 4).

Fig. 4:	Visualization of RAPD band pattern of 6 individuals O.c. toeantei with OPA primer 1-10

Table 3:	Similarity of O.c. toeantei matrix based on the presence of the DNA-RAPD tape pattern analyzed by the UPGMA method
♂: Male, ♀: Female)

The results of the calculation of the appearance of DNA bands from the visualization of DNA band profiles (Fig. 4) can be analyzed the similarity of the matrix O.c. toeantei with the UPGMA method as in Table 3.

In the highest value of matrix similarity (coefficient similarity) is 0.767 at altitude of 140 masl.

Fig. 5:	Dendrogram data of 8 individuals O.c. toeantei based on the presence of DNA-RAPD band patterns analyzed by UPGMA method

This implies that there is a high similarity between O.c. toeantei ♂ and O.c. toeantei ♀ based on RAPD analysis. The lowest value of matrix similarity is 0.517 at altitude of 140 masl and 600 masl. This, implies that the DNA appearance on the RAPD analysis has the lowest similarity, in which for O.c. toeantei ♀ at the altitude of 140 masl and for O.c. toeantei ♀ at the altitude of 600 masl. The dendrogram resulted from average matrix similarity 10 primer OPA can be seen on the Fig. 5.

The result from UPGMA (dendrogram) analysis showed that on the similarity value of 0.57, 2 main cluster of O.c. toeantei at the altitudes of 140 masl, 360 masl and 600 masl were formed. The I main cluster with the similarity value of 0.63 formed into 2 sub clusters consisting of 1 main sub cluster with similarity value of 0.72 that are O.c. toeantei ♀ 360 masl and O.c. toeantei ♂ 360 masl. 2 main sub clusters with similarity value of 0.77 that are O.c. toeantei ♀ 140 masl and O.c. toeantei ♂ 140 masl. Thus, it is known that the more character similarity in morphology is found on O.c. toeantei at the altitude of 140 masl and the lowest similarity at the altitude of 360 masl.

DISCUSSION

The result of this study showed that there are similarity on the clustering pattern of morphological and molecular-RAPD character dendrograms of O.c. toeantei at different level of altitude on Morotai Island. The most high similarity was found at the location of 140 masl of altitude (Table 2). This, became the evidence that morphological variability of the O.c. toeantei butterfly can be influenced by the different of altitude. The sex on the other hand, did not shows any influence to the variation of morphometry and molecular-RAPD, however its only visible on color's variation at the body and wings. The result of this investigation also showed that the Morotai Island has the high categorize of O.c. toeantei intraspecies variations. It can be seen from its lowest value of similarity in both morphometry and molecular-RAPD (Table 1 and 3). Furthermore, from the cluster UPMGA analysis (Fig. 2 and 3) its been known that the similarity value between the main cluster were 0.56 on the morphometry and 0.56 on the molecular-RAPD. This shows that the similarity between cluster is low at morphological as well as molecular-RAPD.

The similarity of morphological and molecular-RAPD character that appears at certain level of altitude indicates the different habitat of O.c. toeantei. One of the main factor of this clustering pattern is the food abundance (mussaenda and ashoka). This is in line with the study of Koneri and Saroyo¹⁷ who stated that based on the type of the butterfly, the variation and abundance of the species is tend to be high at low part than the high part of the area which related to availability of the food. Moreover²⁴ stated that the amount of food at the high level of area was very limited even none for Ornithoptera spp., while at the lower part have the high amount of it Van Vu and Vu³², Van Lien and Yuan³³ and Dendang³⁴ stated that the more high the variety of vegetation the more high the variety of butterfly.

The result from UPGMA on the morphological and molecular-RAPD dendrograms (Fig. 4 and 5) explains that each dendrogram formed into 2 main cluster which organized at each level of altitude. The main similarity value of morphological character dendrogram is 0.56 which also the same at molecular-RAPD character dendrogram. This data showed an in line condition at the morphological and molecular-RAPD characterization. The similarity value of O.c. toeantei an endemic butterfly of Morotai Island falls into low category which below 80%³⁵. The low of similarity values implies the high of diversity. The similar study by Mas’ud²⁴ on the genetic diversity of Ornithoptera croesus reported that the genetic diversity is correlated positively to the high of hotspot level. Variational genetic studies of two Cirrochroa using the RAPD-PCR technique conducted by Zothansangi et al.²¹, showed that there are genetic variations with the similarity of the two cryptic species. The RAPD technique that can determine molecular characterization which has morphological similarities to four morphologically similar butterflies similar to Pieridae²⁰.

Dendrogram construction provides the evidence that intraspecific variation (genetic variation) at the molecular-RAPD level between O.c. toeantei ecotype. The dendrogram results (Fig. 4) based on molecular-RAPD data shows a number similarities with the morphological differentiation of 2 main clusters and 2 subcluster of O.c. toeantei from different level of altitude. The clustering pattern of the 2 main clusters is supported by morphological similarities of 2 subclusters. The main cluster and subcluster patterns strongly support morphological characters. The RAPD technique has revolutionized the field of molecular biology for the study of butterfly DNA, gene flow between populations, evaluation of genetic population structures, determination of genetic relationships and phylogenetics. Likewise in the study of genetic diversity of O.c. toeantei butterfly an endemic from Morotai Island.

This research is the first attempt to conserve endemic butterflies on the Morotai Island of North Maluku carried out at the level of intraspecies diversity of O.c. toeantei based on morphological and molecular characters. This research was also carried out as an effort to preserve the endemic butterfly of North Maluku, Indonesia. Butterfly is a genetic resource that needs to be preserved as a pollinator and biological wealth which has ecological and economic value.

CONCLUSION

There is a similarity in the clustering pattern between UPGMA analysis on morphometric and molecular-RPAD characters of O.c. toeantei the endemic butterfly of Morotai Island. The main similarity values of the UPGMA analysis were 0.56 in the morphometric dendrogram and 0.56 in the molecular-RAPD dendrogram. This information shows that the O.c. toeantei has a high similarity on the habitat with the same altitude, whereas in habitats with different altitude the similarity tends to low. In general, O.c. toeantei the endemic butterflies of Morotai has a high genetic diversity at the intraspecies level.

ACKNOWLEDGMENT

The author would like to thank Ms. Susiati, S.Si, M. BioMed as the head of the Laboratory of Molecular and Cellular Biology at Brawijaya University Malang who accompanied the researchers in the Lab. Mrs. Djunijanty Peggy, M.Sc. PhD as LIPI butterfly researcher who has provided literature information to researchers.