Bulletin of Entomological Research, Page 1 of 6 © Cambridge University Press 2015
doi:10.1017/S0007485315000358
DNA barcoding implicates 23 species and four orders as potential pollinators of Chinese knotweed (Persicaria chinensis) in Peninsular Malaysia M.-M. Wong1,2, C.-L. Lim1,3 and J.-J. Wilson1,4* 1Ecology
and Biodiversity Program, Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia: 2Institute of Plant and Microbial Biology, Academia Sinica, Taipei 11529, Taiwan: 3Herbarium, Forest Research Institute Malaysia, 52109 Kepong, Selangor, Malaysia: 4Museum of Zoology, Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia Abstract Chinese knotweed (Persicaria chinensis) is of ecological and economic importance as a high-risk invasive species and a traditional medicinal herb. However, the insects associated with P. chinensis pollination have received scant attention. As a widespread invasive plant we would expect P. chinensis to be associated with a diverse group of insect pollinators, but lack of taxonomic identification capacity is an impediment to confirm this expectation. In the present study we aimed to elucidate the insect pollinators of P. chinensis in peninsular Malaysia using DNA barcoding. Forty flower visitors, representing the range of morphological diversity observed, were captured at flowers at Ulu Kali, Pahang, Malaysia. Using Automated Barcode Gap Discovery, 17 morphospecies were assigned to 23 species representing at least ten families and four orders. Using the DNA barcode library (BOLD) 30% of the species could be assigned a species name, and 70% could be assigned a genus name. The insects visiting P. chinensis were broadly similar to those previously reported as visiting Persicaria japonica, including honey bees (Apis), droneflies (Eristalis), blowflies (Lucilia) and potter wasps (Eumedes), but also included thrips and ants. Keywords: ants, bees, DNA barcoding, Malaysia, thrips (Accepted 22 March 2015)
Introduction Persicaria chinensis (Polygonaceae) is a perennial herb native to tropical and subtropical eastern Asia. The plant can tolerate a wide range of environmental conditions, including shade, high temperatures, high salinity and drought and is abundant in wet valleys and on grassy slopes in China, from sea level to elevations of 3000 m (Galloway & Lepper, 2010). In peninsular Malaysia, P. chinensis is frequently found growing
*Author for Correspondence Phone: +603-7967 4112 Fax: +603-7967 4187 E-mail:
[email protected]
wild in montane forests up to 1200 m and flowers year-round. It is also cultivated in the lowlands by the ethnic Chinese community who use the herb in traditional medicine to treat lung ailments. The cultivated plants rarely set fruit and are propagated through cuttings. P. chinensis has been categorized as a ‘high risk’ invasive species in the Pacific, based on a comprehensive risk assessment involving 41 criteria (PIER, 2010), and was recently discovered in New Zealand, with the traditional medicine trade implicated as a factor in this incursion (Galloway & Lepper, 2010). Besides ecological and economic importance as a rapid colonizer and a medicinal herb, P. chinensis is of evolutionary interest as a classic example of heterostyly (flower and pollen dimorphism) (Reddy et al., 1977). Heterostyly, together with the production of showy inflorescences (fig. 1), nectar and a
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Fig. 1. Growing habit of Persicaria chinensis and a potential pollinator visiting an inflorescence at Ulu Kali, Pahang, Malaysia.
strong scent, suggests that the wild plant is extensively crosspollinated by animal vectors. We have observed that open pollination resulted in three times more fruits than selfpollination (Wong, 2012). However, the insects associated with P. chinensis pollination have received little attention. A survey of social bee and food plant associations in India identified Apis cerana as a visitor to P. chinensis flowers (Thomas et al., 2009), while a study in Japan identified 15 morphospecies as potential pollinators of the congeneric, Persicaria japonica (Nishihiro & Washitani, 1998). A ‘key question’ in pollination biology (Mayer et al., 2011) concerns the factors determining the diversity of insect pollinators, but lack of taxonomic identification capacity is an impediment to research in this area (FAO, 2009). DNA barcoding, the use of a short standardized mtDNA sequence for species identification (Hebert et al., 2003; Hebert & Gregory, 2005; Floyd et al., 2009), has been suggested as a solution for the taxonomic impediment to pollinator studies (FAO, 2009). In the present study we aimed to elucidate the insect pollinators of P. chinensis in peninsular Malaysia using DNA barcoding. Invasive plants generally lack specialized pollinator requirements (Bartomeus et al., 2008). Therefore, as a widespread invasive plant we would expect P. chinensis to be associated with a diverse group of generalist insect pollinators.
Materials and methods Wild P. chinensis is a scrambling herb found in isolated patches, termed ‘populations’, but which may be a single organism, covering an area of ground 4–10 m2 and growing up to 3 m vertically (fig. 1). We conducted observations on three populations of P. chinensis in the montane forest of Ulu Kali, Pahang, Malaysia (fig. 2), on alternate days between 16 May 2012 and 2 June 2012, from 0600 to 1400 each day. We divided our time equally between the three populations, which all were growing in direct sunlight along the roadside. Insects were observed to move between different inflorescences in the population. The frequency of visits to P. chinensis flowers by
different insect morphospecies was assessed qualitatively with a morphospecies, for the purpose of this study, comprising individuals which were very difficult to tell apart during our field observations. Forty flower visitors, representing the range of morphological diversity observed, were captured in the proximity of inflorescences using a sweep net. The collected insects were observed under a dissecting microscope for the presence of pollen grains adhered to their bodies. Any pollen grains found were mounted on slides using glycerine jelly, counted and examined under a light microscope to determine if they were P. chinensis. A leg was removed from each insect specimen for DNA barcode generation using standard methods (Wilson, 2012). In brief, DNA was extracted using glass-fibre plates and a fragment of COI mtDNA amplified using the ‘Lep’ primer combinations mentioned in Wilson (2012). Cycle sequencing was performed bi-directionally using the PCR primers. The DNA barcodes were uploaded to the Barcode of Life Datasystems (BOLD) (http://www.boldsystems.org; Ratnasingham & Hebert 2007). Upon upload to BOLD the DNA barcodes (>500 bp) were automatically assigned Barcode Index Numbers (BINs; Ratnasingham & Hebert, 2013). BINs are Molecular Operational Taxonomic Units (MOTU) produced by Refined Single Linkage (RESL) analysis across the BOLD database and have been shown to correspond closely with traditional species limits characterized by morphology (Ratnasingham & Hebert, 2013; Hausmann et al., 2013). In addition to RESL, the DNA barcodes were arranged into MOTU using two other approaches commonly applied to DNA barcode data (Boykin et al., 2012) that do not require a priori assignment of DNA barcodes to groups. The Automated Barcode Gap Discovery (ABGD; Puillandre et al., 2012) web interface was accessed from http://wwwabi.snv.jussieu.fr/public/abgd/ abgdweb.html. Previous studies have shown that there is typically a distinct pattern to intra- and interspecies DNA barcode divergences, a so-called ‘barcode gap’, but that this pattern can be unique to a dataset. ABGD uses an automatic recursive procedure to converge on the best pattern for the dataset and
DNA barcoding implicates 23 species and four orders as potential pollinators of Chinese knotweed
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Fig. 2. The location of the three study populations at Ulu Kali, Pahang, Malaysia (03°25′55″N, 101°47′05″E; 03°24′55″N, 101°47′02″E; 03°24′ 45″N, 101°47′22″E).
arranges DNA barcodes into species accordingly (Puillandre et al., 2012). The method has been shown to perform well with sympatric datasets, is fast and user-friendly and does not have any special computational requirements (Paz & Crawford, 2012). A Bayesian implementation of the Poisson Tree Processes (bPTP; Zhang et al., 2013) model was accessed through the web interface available at http://species.h-its. org/ptp/. bPTP can be used to delimit phylogenetic species in a similar way to the popular and widely used General Mixed Yule Coalescent (GMYC) approach (Pons et al., 2006), but without the requirement for an ultrametric tree (Zhang et al., 2013). The DNA barcode dataset was collapsed to unique haplotypes and a Maximum-Likelihood tree was generated in MEGA6 (Tamura et al., 2013) using the ‘best’ model (GTR + G + I), Subtree-Pruning–Regrafting – Extensive (SPR level 5) and otherwise default settings for input into bPTP. Representatives of each MOTU were submitted to the BOLD identification engine full database (‘Full DB’) to assign a taxonomic name to the MOTU. Species names were assigned using a >98% sequence similarity threshold. When there was no match >98%, the ‘Tree-Based Identification’ option was followed on BOLD and higher taxon names were assigned using tree-based criteria following Wilson et al. (2011; table 1).
Results The DNA barcodes generated for this study, photographs and metadata of the specimens are available on BOLD in public dataset: DS-POCK (doi: dx.doi.org/10.5883/DS-POCK; GenBank Accessions KF200036-KF200075). RESL and ABGD converged on the same MOTU (fig. 3), indicating 23 species had been observed visiting P. chinensis flowers. bPTP indicated the sample comprised 20 species. Of the 23 putative taxa, six could be assigned a Linnaean species name, and one could be assigned a ‘dark taxa’ species name (Prenolepis
sp. MAL01) (table 1). Dark taxa are previously recognized species, which have not been provided with a formal name, but which are nevertheless present on taxonomic databases such as BOLD (Maddison et al., 2012; Wilson et al., 2014). Using treebased criteria, nine of the remaining species could be assigned to genus, three to family, and three to order (table 1). One coleopteran species could not be assigned at any level using DNA barcoding and the closest matching sequence was a moth. We subsequently were able to note that insects from the genera, Apis, Askarina and Prenolepis, were the most frequent visitors to P. chinensis flowers. Under a dissecting microscope we observed that all of the insects, with the exception of the hemipterans, had P. chinensis pollen grains adhered to their bodies (table 1). Pollen grains from other plants were also observed on some specimens but the plant species were not identified.
Discussion DNA barcoding implicated a diverse array of insects as potential pollinators of Chinese knotweed in peninsular Malaysia. Using RESL and ABGD, 17 morphospecies were assigned to 23 species representing at least ten families and four orders. Using the DNA barcode library (BOLD) 30% of the species could be assigned a species name, and 70% could be assigned a genus name. Failure of DNA barcodes to match to their closest relative in the library is analogous to ‘longbranch attraction’ in molecular systematics, and can be overcome through expanding taxonomic coverage in the library. Beetles, unlike bees and moths, have not yet been the focus of a global DNA barcoding campaign (but see Woodcock et al., 2013; Pentinsaari et al., 2014 and Hendrich et al., 2014 for recent work from Europe and North America), which could explain the case where a beetle matched most closely (although only sharing 85% similarity) to a moth DNA
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Table 1. Taxonomic assignment of 23 species visiting flowers of Persicaria chinensis at Ulu Kali, Pahang, Malaysia. ABGD group (BIN)1
n
Taxonomic assignment
Assignment criterion
Closest BOLD match (K2P distance)
P. chinensis pollen2
1 (ACF2414) 2 (AAB2866) 3 (ACG0992) 4 (ABX8834) 5 (ACG0411) 6 (ACG1100) 7 (ACQ1337) 8 (AAA5667) 9 (ACG0947) 10 (ABX9044) 11 (ACG0324) 12 (ACG0353) 13 (ACG0325) 14 (ACG0991) 15 (AAC9294) 16 (ACG0063) 17 (ACD7381) 18 (n/a) 19 (ACG0265) 20 (n/a) 21 (n/a) 22 (AAA8457) 23 (ABX7554)
7 1 1 2 1 1 1 1 1 3 1 1 1 1 2 2 1 1 1 1 1 5 3
Diptera: Syrphidae: Episyphus balteatus Diptera: Syrphidae: melanostoma mellinum Diptera: Syrphidae: Asarkina Diptera: Syrphidae: Asarkina Diptera: Syrphidae: Asarkina Diptera: Syrphidae: Eristalinus Diptera: Calliphoridae: Lucilia porphyrina Diptera: Calliphoridae: Chrysomya megacephala Diptera: Muscidae: Limnophora Diptera: Muscidae: Limnophora Diptera: Muscidae Diptera: Muscidae Diptera: Muscidae Hemiptera: Miridae Hemiptera: Cicadellidae: Balclutha incisa Hymenoptera: Vespidae: Eumenes Hymenoptera: Formicidae: Prenolepis sp.MAL01 Hymenoptera: Formicidae: Prenolepis Coleoptera Coleoptera Hymenoptera: Colletidae: Hylaeus Hymenoptera: Apidae: Apis cerana Thysanoptera: Thrypidae: Taeniothrips
Match >98% Match >98% Strict tree-based Strict tree-based Strict tree-based Strict tree-based Match >98% Match >98% Strict tree-based Liberal tree-based Liberal tree-based Liberal tree-based Strict tree-based Strict tree-based Match >98% Strict tree-based Match >98% Strict tree-based Morphology Liberal tree-based Strict tree-based Match >98% Liberal tree-based
Episyphus balteatus (100%) Diptera (98%) Asarkina (97%) Asarkina porcina (97%) Asarkina porcina (95%) Eristalinus (96%) Lucilia porphyrina (100%) Chrysomya megacephala (100%) Limnophora triangula (90%) Diptera (90%) Heliographa ceylanica (90%) Myospila lauta (95%) Myospila lauta (92%) Neolygus hirticulus (92%) Balclutha incise (100%) Hymenoptera (96%) Prenolepis sp.MAL01 (100%) Prenolepis sp.MAL01 (91%) Pyralidae (85%) Coleoptera (84%) Hylaeus setosifrons (89%) Apis cerana (99%) Taeniothrips major (85%)
+ + + + + + + + + + + + + − − + + + + + + ++ +/−
1
Some short DNA barcodes (100 grains observed per specimen; +/−, 0–20 grains observed per specimen; −, no grains were observed.
2
barcode and demonstrates why similarity alone cannot be used for taxonomic identification in the absence of species-level matches (Wilson et al., 2011). Although overall the assignment rate may seem low, the species have the potential to be assigned more precisely in the future as the DNA barcode library continues to grow. Presently, cosmopolitan species are easily identified with DNA barcoding but a focus on regional libraries will be required to precisely assign most specimens a species name. However, standardized DNA barcoding (and the BIN) is potentially more useful at facilitating taxonomic connections between studies than morphospecies names such as ‘Unidentified Syrphidae sp. 1’. Our results are broadly similar to those of Nishihiro & Washitani (1998) who recorded 15 morphospecies, from three orders (Hymenoptera, Diptera and Lepidoptera), visiting flowers of P. japonica. We did not observe any lepidopterans but did observe species from the orders Hemiptera, Coleoptera and Thysanoptera besides Hymenoptera and Diptera. Like Nishihiro & Washitani (1998) we also found the insect visitors comprised honey bees (Apis), droneflies (Eristalis), blowflies (Lucilia) and potter wasps (Eumedes). During our study, we observed that honey bees (Apis cerana) were the most common visitors to P. chinensis flowers, and among all the insects collected, had the largest number of the pollen grains adhered to their bodies. Given the high frequency of visits, and greatest pollen load, A. cerana is probably the most important pollinator of P. chinensis. Thrips, assigned to Taniothrips (Thripidae), were common visitors to P. chinensis and were abundant in inflorescences. Their foraging behaviour was similar to that seen at Antigonon leptopus (Polygonaceae) (Raju et al., 2001), mostly moving within one flower and occasionally moving to adjacent flowers of the
same inflorescence. Thrips have been suggested as potential pollinators of many economically important plants, but besides playing this beneficial role, they are also known to cause extensive flower damage (Tillekaratne et al., 2011). Ants, assigned to Prenolepis (Formicidae), were also among the most frequent insect visitors to P. chinensis flowers. Typically, ants have been considered ineffective pollinators due to their small size preventing effective contact with anthers and stigmas, and a tendency to destroy ovaries during nectar foraging (Gomez & Zamora, 1992). Ants have been suggested as pollinators of Polygonum cascadense (Polygonaceae) (Hickman, 1974) and we found that ants visiting P. chinensis were carrying P. chinensis pollen grains on their thorax, abdomen and legs. Further studies are needed to determine if these ants are effective pollinators or merely nectar thieves. The results confirm our expectation that a taxonomically diverse group of insects are visiting the flowers of P. chinensis in peninsular Malaysia. Although we observed P. chinensis pollen grains adhered to the bodies of most of the captured insects, further surveillance programmes are required to confirm the level of pollination service provided by each of these species. In general, a lack of pollinators does not appear to be a factor limiting the spread of invasive plants (Richardson et al., 2000; Bartomeus et al., 2008) and our study suggests this is the case for P. chinensis. How the presence of invasive P. chinensis plants would affect pollination of native plant species, which may rely on the services of the same group of generalist pollinators, is an area requiring further study. Such studies will undoubtedly benefit from new genomic approaches to resolving plant–pollinator interactions (Clare et al., 2013). Uncovering the diversity, and identities, of flower visitors is a critical first step.
DNA barcoding implicates 23 species and four orders as potential pollinators of Chinese knotweed
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Fig. 3. BOLD Taxon ID tree (Kimura 2 parameter, neighbour-joining) showing the ABGD groups and BIN assignments. Taxon labels include taxonomic assignment, BOLD process ID, sequence length, family assignment and BIN.
Acknowledgements MMW was supported by a University of Malaya Postgraduate Fellowship. The research work was supported by the University of Malaya Research Grant RP003D-13SUS to JJW.
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