Sexual dimorphism is often directly linked to sexual selection, mating systems and resource partitioning, which are crucial in species conservation and management. Many avian species, including pollinator birds, are sexually dimorphic with respect to size and colour, yet, such differences may be subtle for some species. In this study, molecular sexing was performed in addition to determining morphological parameters that can aid in future sex determination of a common forest pollinator, the little spiderhunter (Arachnothera longirostra), in Peninsular Malaysia. Based on 23 captures made in four forests, two out of seven body measurements (i.e. wing and tail lengths) were useful in predicting the sexes of the bird with 100% accuracy. In addition, significant differences were found in the head, bill, and total body lengths. Such findings will facilitate more effective sex identification in future field studies, particularly in the case of juveniles

Abdl Jalil, Norehan

Ng, Wei Lun

Puan, Chong Leong

Yong, Christina Seok Yien

Universiti Putra Malaysia Institutional Repository

Pertanika J. Trop. Agric. Sci. 41 (1): 333 - 340 (2018)ISSN: 1511-3701    © Universiti Putra Malaysia PressTROPICAL AGRICULTURAL SCIENCEJournal homepage: http://www.pertanika.upm.edu.my/E-mail addresses: chongleong@upm.edu.my;clpuan@yahoo.com (Chong Leong Puan),ng.wl85@gmail.com (Wei Lun Ng),chrisyong@upm.edu.my (Christina S.Y. Yong),raehan_jalil@yahoo.com.my (Abdl Jalil Norehan) * Corresponding authorArticle history:Received: 06 April 2017Accepted: 06 June 2017ARTICLE INFOMorphometric Sexing of Little Spiderhunter (Arachnothera longirostra) in Peninsular MalaysiaChong Leong Puan1,2,3*, Wei Lun Ng4, Christina S.Y. Yong5 and Abdl Jalil Norehan1 1Faculty of Forestry, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia 2Institute of Tropical Forestry and Forest Products (INTROP), Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia 3Biodiversity Unit, Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia 4School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275 Guangdong, China 5Department of Biology, Faculty of Science, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia ABSTRACTSexual dimorphism is often directly linked to sexual selection, mating systems and resource partitioning, which are crucial in species conservation and management. Many avian species, including pollinator birds, are sexually dimorphic with respect to size and colour, yet, such differences may be subtle for some species. In this study, molecular sexing was performed in addition to determining morphological parameters that can aid in future sex determination of a common forest pollinator, the little spiderhunter (Arachnothera longirostra), in Peninsular Malaysia. Based on 23 captures made in four forests, two out of seven body measurements (i.e. wing and tail lengths) were useful in predicting the sexes of the bird with 100% accuracy. In addition, significant differences were found in the head, bill, and total body lengths. Such findings will facilitate more effective sex identification in future field studies, particularly in the case of juveniles. Keywords: Arachnothera longirostra, Discriminant function analysis, Morphometric sexing, Pollinators, Sexual dimorphismINTRODUCTIONGlobally, three bird families, namely Trochilidae, Meliphagidae and Nectariniidae, are known as pollinators (Cronk & Ojeda, 2008). These families maintain key ecological functions and services whether in the natural or agricultural ecosystems (Ollerton, et al., Chong Leong Puan, Wei Lun Ng, Christina S.Y. Yong and Abdl Jalil Norehan334 Pertanika J. Trop. Agric. Sci. 41 (1): 333 - 340 (2018)2011). Spiderhunters (Nectariniidae) are morphologically distinctive with their long decurved bill. Besides small arthropods, many spiderhunter species consume nectar and hence, also serve as pollinators (Yumoto et al., 1997; Momose et al., 1998; Sakai, et al., 1999; Phillipps & Phillipps 2011; Sakai, et al., 2013), which often exhibit trap-lining behaviour. Unlike the confamilial sunbirds that are often sexually dimorphic with males being relatively more colourful than females, it may be difficult to differentiate the sexes of spiderhunters, unless through careful examination of their body size and pectoral tufts (Cheke et al., 2001). Malaysia has a total of 10 spiderhunter species with the majority of species living in wooded habitats (MNS-BCC 2015). This includes two Bornean endemics (Whitehead’s spiderhunter Arachnothera juliae and Bornean spiderhunter A. everetti) as well as the recently included of the purple-naped spiderhunter (A. hypogrammicum; Moyle et al. 2011) which was once treated as a sunbird. The presence of a relatively high diversity of spiderhunter species, some of which are sympatric, poses more intricate questions with regard to interspecific segregation of niches (Collins, 2008), intersexual partitioning of resources (Paton & Collins, 1989; Temeles & Roberts, 1993) as well as mate choice of these species that are confined to the Oriental region. In addition, knowing the sexes of these pollinating birds may have implications on the understanding of key ecological functions (Ollerton, et al., 2011). The little spiderhunter (A. longirostra) is abundant in disturbed and regenerating forests (Rahman et al., 2010; Wells 2010) and is frequently encountered during mist-netting surveys (Wells, 2010; Phillipps & Phillipps, 2011)  compared with other Malaysian spiderhunter species. Male little spiderhunters can be differentiated from females by their larger sizes and the presence of pectoral tufts (Cheke, et al., 2001; Cheke & Mann, 2008; Wells, 2010, but see Jeyarajasingam, 2012), although Wells (2010) noted that the latter feature may develop later than other adult characteristics. In this study,  sexes of randomly caught little spiderhunters from Peninsular Malaysia were determined using molecular methods, before conducting discriminant function analysis on the morphometric measurements obtained from the field. The intention was to develop a way for morphometric sexing of the species for future field studies, eliminating the need for invasive sex determination.MATERIALS AND METHODSSampling Using mist-nets,  we sampled l i t t le spiderhunters from three forest reserves located in Peninsular Malaysia, i.e. the Bintang Hijau (5°26’20”N, 100°55’27”E; Perak state), Sungai Lalang (3°1’29”N, 101°54’49”E; Selangor state), and Panti (1°51’38”N, 103°54’26”E; Johor state) Forest Reserves, as well as an isolated forest patch within the Shah Alam National Morphometric Sexing of Little Spiderhunter335Pertanika J. Trop. Agric. Sci. 41 (1): 333 - 340 (2018)spring balance, in gram. Blood samples were collected by pricking the brachial vein with a sterile 27-gauge needle (Davis 2005), mixed with 100% ethanol and stored at 4°C. All the birds were released after their blood samples were taken.Botanical Garden (3°5’57”N, 101°30’12”E; Selangor), from January to August 2016.U s i n g  a  c a l l i p e r  a n d  a  r u l e r, morphological measurements were taken for the total body, head, wing chord, tarsus, tail, and bill (culmen) lengths, in mm (Figure 1). Weight was measured using a Figure 1. Measurement of (A) wing, (B) tail, (C) tarsus, (D) bill, and (E) head lengths18   331 Figure 1. Measurement of (A) wing, (B) tail, (C) tarsus, (D) bill, and (E) head 332 lengths 333  334  335  336  337  338  339  340  341  342 Molecular sexing Total genomic DNA from blood samples was extracted from 20 µl of blood using the DNeasy Blood and Tissue Kit (Qiagen) based on the manufacturer’s protocol. The extracted DNA samples were stored at -20°C until further analyses.In birds, sex is determined using the ZW sex determinat ion system, in which males are the homogametic sex  (hav ing  two Z-chromosomes) and females are the heterogametic sex (having one each of the Z- and W-chromosomes).  A primer pair  (CHD1F: 5’-TATCGTCAGTTTCCTTTTCAGGT-3’ a n d  C H D 1 R : 5’-CCTTTTATTGATCCATCAAGCCT-3’; Lee et al. 2010) was used to amplify a section of the sex chromosome CHD gene that is present on both the avian W- and Z-chromosomes in differing fragment lengths. The PCR amplifications were performed in 10 μL reactions, each containing approximately 30 ng of genomic DNA as template, 1 μM of each primer, and 5 μL of NEXproTM ePCR 2× master mix (NEX Diagnostics). The PCR reaction profile comprised an initial denaturation of 3 minutes at 95°C; followed by 30 cycles of 30 sec at 95°C, 30 sec at 50°C, and 2 min at 72°C; and finally, an extension step at 72°C for 7 min. The PCR amplicons Chong Leong Puan, Wei Lun Ng, Christina S.Y. Yong and Abdl Jalil Norehan336 Pertanika J. Trop. Agric. Sci. 41 (1): 333 - 340 (2018)were analysed by electrophoresis on 1.0% (weight/volume) agarose gel, stained with ethidium bromide and viewed under UV illumination. Males would present one DNA band (at ~500bp), while females would present two DNA bands (at ~300bp and ~500bp), on the gels.Morphological data analysesA Mann-Whitney U test was performed to compare differences in the measurements between male and female birds. All body measurements were reported in means and standard errors. Discriminant Function Analysis (DFA) with a stepwise procedure was applied on the measurements. All statistical tests were performed using SPSS Version 16.0 (SPSS, Inc., Chicago, Illinois).RESULTS AND DISCUSSIONA total of 23 little spiderhunters were caught, including 14 from Sungai Lalang Forest Reserve, four from Bintang Hijau Forest Reserve, two from Panti Forest Reserve and three from Shah Alam National Botanical Garden. Eleven (47.8%) were males, as determined through DNA analysis (Figure 2). Visually, we successfully identified eight males (72.7% of the total males caught) through the presence of pectoral tufts. Of the three males which sex was difficult to identify in the field, there was only one juvenile with noticeable orange-yellow gape flange.Figure 2. Molecular sexing results. Males show 1 band (ZZ) at ~500bp, and females show 2 bands (ZW) at ~300bp and 500bp19   343  344 Figure 2. Molecular sexing results. Males show 1 band (ZZ) at ~500bp, and 345 females show 2 bands (ZW) at ~300bp and 500bp 346  347  348  349  350  351  352  353  354  355  356  357 Significant differences were found in the head, bill, wing, tail, and total body lengths (p ≤ 0.01; Table 1), but not in the tarsus length and weight. Based on DFA, a parsimonious model comprising wing and tail lengths as predictors provided the best possible prediction of the sex of a little spiderhunter with 100% accuracy (Figure 3). The discriminant function (D; Wilk’s Lambda = 0.087, χ2 = 48.785, P < 0.001) was:[D = 0.852*Wing length + 0.531*Tail length]Based on centroids derived from DFA, a bird with a score on the DF closer to 3.580 would be a male whereas it was -2.983 for females. Morphometric Sexing of Little Spiderhunter337Pertanika J. Trop. Agric. Sci. 41 (1): 333 - 340 (2018)In this study, we obtained almost equal sample sizes for both sexes and all body measurements were similar to those reported in literature (Cheke et al., 2001; Cheke & Mann, 2008; Wells, 2010; Jeyarajasingam, 2012). Our results indicated that the little spiderhunter can be sexed based on five morphological parameters. Albeit not significantly heavier, male birds have longer bodies, wings, tails, bills, and heads. Despite the small sample size, the study has also demonstrated that pectoral tufts may be a good indication of sex i.e. adult male. However, such characteristics may not be observed in juveniles and subadults that do not have visible orange-yellow gape flange (Wells, 2010). Hence, for subadults and juveniles, wing and tail lengths may be useful to discriminate the sexes. Figure 3. Scatterplot of wing length against tail length measured from the little spiderhunter showing a clear separation according to sexes (n = 23)20    358 Figure 3. Scatterplot of wing length against tail length measured from the little 359 spiderhunter showing a clear separation according to sexes (n = 23) 360  361  362  363  364  365  366  367 Table 1 Morphometric measurements from little spiderhunter and results of Mann-Whitney U-test Measurements Male (n = 11) Female (n = 12) Z p ± SE 95% CI  ± SE 95% CIHead length (mm) 60.09 ± 3.62 51.51-69.49 52.75 ± 0.54 51.57-53.93 -3.721 <0.001Bill length (mm) 38.27 ± 0.88 35.92-40.08 34.25 ± 0.54 33.07-35.43 -3.382 0.001Wing length (mm) 68.18 ± 0.33 67.33-68.67 60.17 ± 0.49 59.09-61.25 -4.097 <0.001Tail length (mm) 45.18 ± 0.55 43.80-45.80 38.71 ± 0.59 37.42-40.00 -4.101 <0.001Tarsus length (mm) 20.20 ± 0.13 19.90-20.50 19.25 ± 0.46 18.23-20.27 -2.458 0.014Total body length (mm)152.27 ± 2.49 145.80-158.20 142.50 ± 1.98 138.14-146.86 -2.994 0.003Weight (g) 13.18 ± 0.52 11.90-14.50 12.75 ± 1.72 8.97-16.53 -1.686 0.092Chong Leong Puan, Wei Lun Ng, Christina S.Y. Yong and Abdl Jalil Norehan338 Pertanika J. Trop. Agric. Sci. 41 (1): 333 - 340 (2018)Being frequently encountered during mist-netting sessions, and being the most widespread among its congeneric counterparts, with specialised feeding structure, the little spiderhunter has great potential as a focal species for the study of complex ecological interactions in the tropics (Olsen et al., 2013). For example, since male little spiderhunters have longer bills than the females, it would be interesting to examine if differential foraging is present between the sexes, i.e. possible interactions between the birds, as pollinators, and floral anatomy (Paton & Collins, 1989; Sakai et al., 1999; Cronk & Ojeda, 2008; Sakai et. al., 2013). Such research is especially important in view of the increasing risk of losing avian pollinators around the world (Regan et al., 2015).CONCLUSION Using morphometric data collected in the field, and supported by molecular sex determination, we were able to evaluate the feasibility of morphometric sex determination in the little spiderhunter in Peninsular Malaysia. Since the sexing of birds is important in behavioural and ecological studies, this study provided an inexpensive, rapid, and accurate way for future sex determination of the little spiderhunter. ACKNOWLEDGMENTSThis research was funded by Universiti Putra Malaysia through the Putra Grant (Project No. GP-IPS/2015/9468400) and partly by the Tan Kean Cheong Bird Conservation Memorial Fund. The authors thank the Forestry Department of Peninsular Malaysia for the permission to access to the forest reserves and the Department of Wildlife and National Parks Malaysia for the permission to carry out bird surveys and blood sampling. We also thank Mohammad Gaddafi Denis, the former director of the Shah Alam National Botanical Garden for providing access and accommodation at the botanical garden. This research was carried out under the approval given by the Institutional Animal Care and Use Committee, Universiti Putra Malaysia (UPM/IACUC/AUP-R081/2015). We are grateful to all those who assisted us in the field. Fong Chun Wah has kindly produced the illustration needed in this manuscript.REFERENCES Cheke, R., & Mann, C. (2008). Family Nectariniidae (Sunbirds). In  J. del Hoyo, A. Elliot & D. A. Christie (Eds.), Handbook of the Birds of the World (Volume 13): Penduline-tits to Shrikes. (p. 196–321). Barcelona: Lynx Edicions. Cheke, R. A., Mann, C. F., & Allen, R. (2001). Sunbirds: A Guide to the Sunbirds, Flowerpeckers, Spiderhunters and Sugarbirds of the World. (p. 384). London: Christopher Helm Publishers.Cronk, Q., & Ojeda, I. (2008). Bird-pollinated flowers in an evolutionary and molecular context. 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Morphometric sexing of little spiderhunter (Arachnothera longirostra) in Peninsular Malaysia

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Morphometric sexing of little spiderhunter (Arachnothera longirostra) in Peninsular Malaysia

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