DISTINGUISHSING SPECIES AMONG MEMBERS OF

CHAPTER V DISTINGUISHSING SPECIES AMONG MEMBERS OF

Himantura uarnak: MORPHOLOGICAL APPROACH Abstact The assessment on morphological and stingray species distribution of Himantura uarnak species complex was based on data collection from August 2006 to October 2011, covering eight sampling sites in the western and eastern Indonesia. Sample collection for the western region, include: 1 Sunda Strait Banten, 2 Batang Central Java and 3 Baron, south coast of Yogyakarta, while the eastern region, include: 4 Singaraja Bali, 5 Labuan Bajo NTT, 6 Jampea and 7 Makassar south Sulawesi, and 8 Kendari Southeast Sulawesi. Handling sample followed the procedures for the preparation of specimens of the National Museum of Zoology Bogoriense MZB, Cibinong. The specimen was identiified based on the key of Dasyatidae family, for genus Himantura. Spotting pattern of reticulates on the dorsal side of stingray individuals was closely examined. Size measurement included body width cm, weight kg, and length cm. Morphology spotting patterns was also used to determine which of the two cluster 1 or 4 is the true H. leoparda. The spotting patterns on Himantura uarnak species complex were high variation showed that this complex consists of several species. The comparison between spotting patterns and molecular approaches found some inaccuracies, so it was concluded that the morphological approach should not be used to identify the species complex without other approachs. Key words: Morphological character, spotting pattern, Himantura uarnak species complex, stingrays Introduction Complex species is a group of closely related species; the boundaries among species are often unclear or vague and usually show incomplete reproductive isolation Mayr 1992, Irwin et al. 2001; Queiroz 2007. Himantura uarnak complex species are members of Dasyatidae family, the most economical important stingrays in the waters of Indonesia Tull 2009; Fisheries Statistics 2010 and widely used as food and trading products such as handbags, wallets, belts and other accessories Adrim 2008. Members of this complex species Himantura uarnak, H. undulata and H. leoparda are still in debate whether they are different species, and often misidentified. These stingrays are generally identified morphologically especially the spot pattern and body shape. Spotting pattern in Himantura uarnak, H. undulata and H. leoparda differs only when the individuals reach adult stage. Spots in H. uarnak is all over the dorsal bady, winding like a beehive, while in Himantura undulata they resemble leopard spots. Character shares between these species occurs in their body shape which is rectangular widened when they are young, andrectangular plate quadrangular when they reach adult. Himantura leoparda has rhomboidal body shape. Similarities between H. uarnak and H. undulata look at the shape of their bodies that plate when juvenile rectangular widened, while his adulthood rectangular plate quadrangular. Himantura leoparda have characteristics similar to H. undulata, but there is little difference in the characteristic ring of leopard spotting pattern on the dorsal surface. This species also has a subtle difference in squamation and body shape, as well as the development of color has a unique and complex. Aside from that, the body shape of H. leoparda are rhomboidal not quadrangular as in H. uarnak and H. undulata Manjaji 2004; Manjaji-Matsumoto Last 2008. The chapter presents the distribution pattern of Himantura uarnak species complex in Indonesian waters based on morphological characters spotting pattern, species and sex. Materials and Methods Time and place of study Data collection was conducted in August 2006 Arlyza 2006-2008, unpublished to October 2011, covering eight sampling locations in the western and eastern Indonesia. Specimen collection Stingray of Himantura were collected from euther fish landing places production center or gathered from stingray fishing collector. All relevant information coming with each specimen was documented, such as: date of sampling, specimen code, museums code, catchment population, location of catchment, and local name Arlyza 2006-2008, unpublished. The one individual already get a code of museums number, MZB 20875 and still there two more new specimens are yet coded. Specimens were collected only for species that were small and medium size range between 20-45 cm of body width and 10- 20 kg of body weight , while the large size only for documented from all directions and special parts. Each specimen was immersed in 4-7 formalin solution for 2-3 weeks and then re-immersed in a solution of alcohol 70 and deposited in the Museum of Zoology Bogoriense MZB, Cibinong. The specimen collection was used to clarification of the molecular result based on DNA barcoding. Identification of specimens The specimen identification followed the key of Dasyatidae family, genus Himantura Compagno Roberts 1982; Monkolprasit 1984, Nishida and Nakaya 1990; Michael 1993; Last Stevens 1994; Manjaji-Matsumoto Last 2008. Morphological data was recorded by measuring body width W, body length L using a roll meter Figure 5.1, while the body weight BW was measured by using scales balance. Himantura uarnak Female, IR109 H. leoparda Male, IR36 Figure 5.1 The measurement of length and width of Himantura uarnak species complex Body width Body width Body le n ght Body le n ght The individuals of stingrays was sexed based on the presence of clasper for male stingrays Figure 5.2 and Table 5.1. Figure 5.2 Clasper, male genitalia on stingrays Table 5.1 The number of individual on Himantura uarnak species complex based on species and sex Locations Species H. uarnak H. undulata H. leoparda ♂ ♀ ♂ ♀ ♂ ♀ Batang, Jawa Tengah 1 33 51 Selat Sunda, Banten 1 2 Makassar, Sulawesi Selatan 6 5 Jampea, Selayar 2 5 Kendari, Sulawesi Tenggara 1 2 Labuan Bajo, NTT 2 Singaraja-Buleleng, Bali 1 Baron, Yogyakarta 1 Total per sex 11 12 1 34 55 Total individuals 113 23 1 89 spot pattern was observed on on upper side of the stingray body Appendix 15. Himantura uarnak complex species generally have a spot small and large varied size of spots and tended to be similar in their juveniles Figure 5.3a. In adults, the spot pattern changed into Figure 5.3b, honeycomb Figure 5.5a, leopard patterned Figure 5.5b and leopard ringed Figure 5.5b type 2 White et al. 2006 ; Manjaji-Matsumoto Last 2008. a. apopyle b. clasper groove c. pseudosiphon d. hypopyle e. pseudopera Figure 5.3a Juvenile of H. uarnak IR106 Figure 5.3b Adult of H. uarnak IR109 Himantura uarnak species complex also showed another characteristic: H. uarnak has two prominent spiny pearl liyingon the heart and center of the disk-shaped crescent and followed by smaller spines Figure 5.4a. Whereas in H. undulata or H. Leoparda, those two pearls are both larger thorny heart-shaped and followed by small thorns heart- shaped Figure 5.4b Data Analysis Data analysis of the morphological approach just prioritized on spotting pattern comparison with the results of the molecular approach on each individual in H. uarnak species complex. Results and Discussion Spotting patterns of Himantura uarnak species complex Material samples of stingray has successfully collected from 8 sampling locations in Indonesian waters with the number of collection was 113 individuals, consisting of 70 females and 43 males. The spotting pattern of Himantura uarnak species complex was shown in Appendix 14 and Table 5.2. Table 5.2 The number of individual on Himantura uarnak species complex based on spotting patterns and sex Species Spotting Types Total Type11R Type22R Type33R ♂ ♀ ♂ ♀ ♂ ♀ H. uarnak 4 7 4 2 3 3 23 H. undulata 1 + H. leoparda 89 2 3 25 31 5 24 90 Total 6 10 29 33 8 27 113 Note: 29 individuals without photos one individual of H. undulata, 7 individuals of H. leoparda var.1 and 21 individuals of H. leoparda and 17 individuals 3 individuals Figure 5.4a The pearl of H. uarnak IR105 Figure 5.4b The pearl of H. leoparda IR48 of H. leoparda var. 1, 8 individuals of H. uarnak and 6 individuals of H. leoparda are juvenile that were not included in identifying the type of spotting patterns. Type1R IR108 Type2R IR98 Type3R IR99 H. uarnak Figure 5.5a H. leoparda or H. undulata Figure 5.5b Type1 IR30 Type2 IR31 Type3 IR45 Figure 5.5 Variation of reticulated spot pattern on H. uarnak species complex based on the form of reticulation Morphological complex species of H. uarnak has been grouped by type of spotting pattern distribution, while the group species groupings based on molecular approach was using mtDNA by COI marker Appendix 14. According to Appendix 14 and 15, stingrays in Group I still there were found variation of spots character, based on direct observations have found more than 2 different spot characters. This may indicate that in the Group I was still possible to find many difference of spotting pattern within species. Group I has five types of 6 spotting patterns were determined, whereas the other type of spotting was Type3 that according to the results of molecular approaches was H. leoparda var.1. The fifth pattern of spots has an average size of 49.56 cm long, 59.33 cm wide and weighs 8.14 kg. Group II has a length of 84.84 cm, a width of 96.82 cm, weight 30.58 kg with a spotting pattern similar to Type 2 that was H. undulata, but based on the morphological approach included in Group IV. Group III has 3 types of spottings, i.e. Type1, Type 2R, and Type 3R with an average length of 59.85 cm, 71.83 cm wide, and weighs 7.35 kg. According to the results of molecular approaches should consist of Type 1R, Type 2R, and Type 3R. Group IV was H. leoparda with an average length of about 29.90 cm, 34.45 cm wide, and weighs 1.09 kg. This group has four types of spotting pattern consisted of the Type 1, Type 1R, Type 2 and Type 3. Meanwhile, according to the moleculer approach, this group should only have two types of spotting, they were Type 1 and Type 2. This situation was indicated that there has been confusion of results through comparison of spotting patterns by morphological approach. Based on the comparison between morphological and molecular approaches, molecular approaches can answered of problem on the complex species compared to morphological approach. Some things were clearly visible the incorrect results obtained by only comparing morphological characteristics between individuals in species complex. Most of H. leoparda into Type 1 and 2, while Type 3 represented to H. leoparda var.1. The spotting patterns of H. undulata only have one type n=1 and tend to be similar to H. leoparda Type 2 Appendix 15. This was confirmed from the results of genetic studied using nuclear DNA markers, Type 2 IR031 have in common alleles with H. undulata IR007 at three polymorphic intron loci i.e. Cam-3A, Cam-3B and Cam-3C. Meanwhile based on the molecular approach used mitochondrial DNA COI maker, H. undulata has a similarity of 92,1 with Type 2, while according to the maker cyt b has similirity in common at 90. Conclusions 1. The spotting pattern of H. leoparda, H.undulata and H. uarnak were different, and they were grouped into three of types. 2. The spotting patterns of H. leoparda as Type 2, which is typical of Cluster 4=Clade IV=Group IV. Therefore, Cluster 4 is the true H. leoparda, Cluster 1 was an undescribed species new species.

CHAPTER VI GENERAL DISCUSSION