The Utility of Barcodes and Sequencing Techniques*
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PL-ISSN 0015-5497 (print), ISSN 1734-9168 (online) Folia Biologica (Kraków), vol. 62 (2014), No 3 Ó Institute of Systematics and Evolution of Animals, PAS, Kraków, 2014 doi:10.3409/fb62_3.223 A DNA Metabarcoding Study of a Polyphagous Beetle Dietary Diversity: the Utility of Barcodes and Sequencing Techniques* £ukasz KAJTOCH Accepted May 15, 2014 KAJTOCH º. 2014. A DNA metabarcoding study of a polyphagous beetle dietary diversity: the utility of barcodes and sequencing techniques. Folia Biologica (Kraków) 62: 223-234. Recently developed techniques of DNA barcoding and next-generation sequencing (NGS) overcome previous limitations of evaluation of animal diet composition and together are promising method in molecular ecology. The objective was to compare standard ABI Sanger sequencing with new high throughput sequencing (Illumina MiSeq) technique and the two selected plant barcodes (rbcL gene and trnL intron) in terms of the identification of host plant composition for the selected beetle species – the Centricnemus leucogrammus weevil. A comparison of two sequencing techniques showed that NGS (in this case Illumina) gave more exhaustive results than the Sanger method. Moreover, it was proven that a two-locus barcoding systems (rbcL and trnL) is sufficient for host plant identification from DNA isolated from insect bodies, at least at the genus level. A comparison of host plant composition among distant populations revealed that the studied species did not feed uniformly across its range. This probably reveals an ecological adaptation of geographically and genetically isolated populations. These findings, beside broadening basic knowledge on the use of barcoding and sequencing techniques for host plant identifications in insect populations, can have implications for conservation studies and strategies for rare and endangered species. Key words: Molecular ecology; chloroplast DNA; rbcL gene; trnL intron; phytophagy; Centricnemus leucogrammus. £ukasz KAJTOCH, Institute of Systematics and Evolution of Animals, Polish Academy of Sci- ence, S³awkowska 17, 31-049 Kraków, Poland. E-mail: [email protected] The feeding preferences of invertebrates repre- material present in samples (fragmentation, diges- sent a difficult field of research. Most studies have tion) and resolution (possibility to identify plant not afforded detailed knowledge about the host species). Only the DNA method has been proved plants and feeding habitats of herbivorous arthro- to be sufficient for precise identification of host pods which would lead to an understanding of their plants and also for quantitative analysis. This complex evolutionary associations and ecological method takes advantage of the DNA barcoding interactions. Moreover, detailed information on concept for species identification (HEBERT et al. the diet of rare and endangered species might be of 2003; MARYAÑSKA-NADACHOWSKA et al. 2010; special value for designing appropriate conserva- KUBISZ et al. 2012a), employed also in ecological tion strategies and management plans (e.g., and biodiversity studies (VALENTINI et al. 2009b; MARRERO et al. 2004). There are several known TABERLET et al. 2012). Several DNA barcodes methods of evaluation of animal diet composition have been proposed for land plants, either indi- (for review see, e.g., MATHESON et al. 2008 and vidually or in combinations (CHASE et al. 2007; VALENTINI et al. 2009a). These methods use either KRESS &ERICKSON 2007; FAZEKAS et al. 2008; direct observation of foraging insects in nature HOLLINGSWORTH 2009), and a two-locus barcode (e.g., SANDHOLM &PRICE 1962) or in laboratory has been proposed: the rbcL gene together with the conditions (e.g., DIECKMANN 1980), or analysis of matK gene (CBOL 2009). However, these bar- gut contents and faeces (e.g., HOLECHEK et al. codes may not always be used for the identification 1982; JOHNSON &NICOLSON 2001). Most meth- of plant species from gut contents as primers for ods of gut contents and faeces analysis have seri- these markers rarely cover a wide spectrum of ous drawbacks related to the quality of plant plant taxonomic units. On the other hand, one _______________________________________ *Supported by grant UMO-2011/01/B/NZ8/01491 from the Polish National Science Centre. 224 £. KAJTOCH chloroplast intron, trnL (TABERLET et al. 1991, The xerothermic habitats of central and eastern 2007; VALENTINI et al. 2009a), has been success- Europe, which sustain a very high biodiversity of fully used for host plant barcoding in insects, par- plants (DZWONKO &LOSTER 2007) and insects ticularly beetles (JURADO-RIVERA et al. 2009; (LIANA 1987; MAZUR 2001) were selected as the NAVARRO et al. 2010; KUBISZ et al. 2012b; target of this study. Simultaneously, these extrazo- KAJTOCH et al. 2013; KITSON et al. 2013). The nal analogs of Eurasian steppes are seriously above studies mostly focused on comparing the se- threatened as a result of their limited and fragmented quences gathered from the studied species with the distribution, as well as anthropogenic transforma- data available from genetic databases such as Gen- tion and degradation (MICHALIK &ZARZYCKI Bank (http://www.ncbi.nlm.nih.gov/genbank/). 1995; MAZUR &KUBISZ 2000; CREMENE et al. This step can also limit the success of identifica- 2005). In xerothermic habitats, there exist rich as- tion, as not all plant barcodes have a wide coverage semblages of herbivorous beetles, and especially of plant sequences in databases; e.g., the rbcL gene weevils (Curculionidae) (MAZUR 2001) and leaf- has 126,485 hits, the matK gene 82,631 hits, the beetles (Chrysomelidae)(W¥SOWSKA et al. 2006). trnL intron 160,932 hits and trnH-psbA spacer Over the past years, great emphasis has been placed only 17,287 hits (GenBank, state as of July 2013). on understanding the phylogeography and conser- These numbers show that the best choices are the vation genetics of selected xerothermic beetle spe- trnL intron, the rbcL and the matK genes. cies (KAJTOCH 2011; KAJTOCH et al. 2011, 2014; Studies on DNA barcoding of host plants were KUBISZ et al. 2012b). The species that has been first based on ABI (Applied Biosystems Inc.) San- best investigated is the weevil Centricnemus leu- ger sequencing; however, this method has serious cogrammus (GERMAR 1824), whose genetics has limitations as it cannot simultaneously generate been studied on the basis of several markers (both data for many samples and an additional cloning mtDNA and nucDNA) (LACHOWSKA et al. 2006; step is needed for the sequencing of multiple am- KAJTOCH et al. 2009; KAJTOCH 2011), as well as plicons. This has been recently overcome by microsatellites (KAJTOCH et al. 2014). These data next-generation sequencing (NGS) techniques for have shown that C. leucogrammus populations can targeted sequencing studies (HARISMENDY et al. be clustered into several geographically separated 2009; EKBLOM &GALINDO 2011; SHOKRALLA et al. clades. It would be interesting to verify if these dis- 2012). Different NGS techniques have been used tinct geographic and genetic units are also distinct for massive sequencing of environmental samples, ecologically. The species is known to be polyphagous e.g., for host plant studies (TABERLET et al. 2007; (DIECKMANN 1980; MAZUR 2001); however, its SOINONEN et al. 2009; POMPANON et al. 2012). host plants have been identified only via direct ob- servations. These observational data show that it is Most studies using DNA plant barcodes for host leave-feeder as imago, however nothing is known plant identification have been performed on indi- about diet of its larva, which probably feed in the vidual species and populations, or even on single root of herbaceous plants as most Entiminae do. individuals (JURADO-RIVERA et al. 2009; NAVARRO The comprehensive knowledge of the zoogeogra- et al. 2010; JUNILLA et al. 2010; GARCIA-ROBLEDO phy and genetics of this particular species makes it et al. 2013) and on mono- or oligophagous species an excellent subject for the present study. (STAUDACHER et al. 2011; KUBISZ et al. 2012b; KAJTOCH et al. 2013). So far only KITSON et al. (2013) studied polyphagous beetle diet on popula- In this study, the DNA-based approach was used tion level. There are no published studies dealing for host plant identification by combining the plant with host plant identification in a large number of barcoding concept with DNA sequencing tech- invertebrate specimens from different areas, while niques. The objective was to compare standard only large-scale sampling from many individuals Sanger sequencing with a new high throughput se- and from different parts of a species range can lead quencing technique (Illumina MiSeq) in terms of to a comprehensive understanding of the species the identification of host plant composition for the feeding preferences and behavior. Such an ap- selected beetle species – the C. leucogrammus proach is however time- and cost-consuming. It is weevil. Moreover, both techniques were used for not always necessary to study many specimens the estimation of differences in host plant compo- separately to obtain data about the host plant com- sition among four regional groups of this beetle. position of particular populations. It is especially The third aim of this study was to compare the effi- problematic for polyphagous and abundant inver- ciency of host plant identification between the two tebrates. In such cases, the solution is to analyze selected plant barcodes (the rbcL gene and the trnL their diet at the level of populations instead of in- intron). The collected