Uncovering the hidden biodiversity of natural history ...

Zootaxa 4250 (6): 541–559 http://www.mapress.com/j/zt/ Copyright © 2017 Magnolia Press

Article

ISSN 1175-5326 (print edition)

ZOOTAXA

ISSN 1175-5334 (online edition)

https://doi.org/10.11646/zootaxa.4250.6.3 http://zoobank.org/urn:lsid:zoobank.org:pub:FBB4C6AC-9D59-44CF-AFDD-9CF929EBF379

Uncovering the hidden biodiversity of natural history collections: Insights from DNA barcoding and morphological characters of the Neotropical genus Orthocomotis Dognin (Lepidoptera: Tortricidae) JÓZEF RAZOWSKI1, VOLKER PELZ 2 & SEBASTIAN TARCZ1 1

Institute of Systematics and Evolution of Animals, Polish Academy of Sciences, Kraków 31-016, Sławkowska 17, Poland. E-mail: [email protected]; [email protected] 2 Bonnenweg 3, D-503809 Ruppichteroth, Germany.

Abstract We used a 227-bp fragment of the mitochondrial gene cytochrome oxidase I (DNA “barcode”) in conjunction with morphological data to study specimens of the Neotropical genus Orthocomotis Dognin, 1906, acquired from natural history collections. We examined over 20 species of Orthocomotis from 17 localities in Colombia, Ecuador, and Peru. The analysis identified 32 haplotypes among the 62 specimens and found no haplotypes shared among species. The molecular study revealed not only the usefulness of short COI sequences in discriminating among Orthocomotis species but also showed distinctness of four clusters which correspond to those based on morphological (genitalia) characters. Moreover, the molecular results suggest the occurrence of rapid speciation in Orthocomotis. We hypothesize that this may be linked to the great biodiversity of potential host plants in Neotropical ecosystems. Key words: COI mtDNA, genitalia, interspecific relationships, morpho-molecular study, phylogenetic network, species discrimination

Introduction Biodiversity estimation is one of the major and controversial challenges of modern biology, not only because of its contribution to understanding ecological and evolutionary processes, but also from an economic perspective (Bartkowski et al. 2015). It is estimated there are from 5±3 million (Costello et al. 2013) to 8.7±1.3 million (Mora et al. 2011) eukaryotic species on the planet, of which only 1.5–1.6 million have been described and classified (Paz & Crawford 2012). Surveying the biodiversity of the Neotropics is particularly difficult because that region likely contains the greatest number of terrestrial species on earth (Tundisi & Matsumura-Tundisi 2008). The discovery of new species is connected not only with the exploration of unstudied ecosystems, such as tropical rainforests, but also of well-characterized taxa with overlooked cryptic diversity (e.g., Hebert et al. 2004, Beheregaray & Caccone 2007). Another area of discovering “hidden diversity” are natural history collections, which are repositories of crucial information for taxonomy (Price et al. 2015), conservation (Gaubert et al. 2006), and biogeography, as well as evolutionary studies (Lister et al. 2011). Identification based on morphological characters may be tenuous and time consuming (Chapple & Ritchie 2013), usually requiring consultation with an expert. In comparison, the application of data obtained from macromolecules offers more rapid and quantifiable opportunities in systematic and phylogenetic research. Although this concept is not new (Zuckerkandl & Pauling 1965), its standardization through the application of DNA barcoding has not only revolutionized species identification (Hebert et al. 2003, 2004), but also permitted the assignment of specimens of various life stages to a particular species (Karthika et al. 2012, Meiklejohn et al. 2013), and the association of individuals of different sexes to the same di- or polymorphic species (Dabert et al. 2011, Slowik & Blagoev 2012); it also has enabled ecosystem biodiversity assessment (Janzen et al. 2005, Radulovici et al. 2010). DNA barcoding has been used successfully in various group of organisms, including bacteria (e.g., Lebonah et al. 2014), Protista (e.g., Kosakyan et al. 2013), plants (e.g., Li et al. 2015), and animals (e.g., Waugh 2007). Among Accepted by J.W. Brown: 9 Feb. 2017; published: 10 Apr. 2017

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lower taxa, it has been applied to the study of insects (Wilson 2012), including Lepidoptera (Hajibabaei et al. 2006a). In contrast to the limitations associated with DNA degradation in older specimens (Mitchell 2015), barcoding has been applied successfully to analyze material deposited in natural history museums (Hebert et al. 2013, Chambers & Hebert 2016). Barcoding of specimens in natural history collections allows not only the of study material which is difficult or impossible to obtain, but also to match via DNA barcodes specimens identified by taxonomic specialists (Mitchell 2015). Tortricidae are among the most diverse and species rich families of microlepidoptera (Brown 2005, Nieukerken et al. 2011), with over 10,000 described species, many of which are important agricultural pests (Gilligan et al. 2014). However, the application of molecular markers in resolving systematic and evolutionary issues in that family has been rather sparse (Regier et al. 2012). Most studies using molecular markers have focused on economically important species (e.g., Schroeder & Degen 2008, Timm et al. 2010). In the present study, a combination of morphological and molecular data allowed us to verify as completely as possible interspecific relationships in Orthocomotis Dognin, 1906, a large genus of Neotropical Tortricidae. Orthocomotis comprises 57 described species distributed across Central and South America. Its systematic position within Tortricidae has been re-evaluated several times (Razowski 1982, Powell 1986, Brown 1989), but the genus is now rather convincingly included in the tribe Euliini (Razowski 2008, Razowski el al. 2013). So far, there has been no hypothesis regarding the infrageneric relationships of species, and the classification has been based only on morphological characters. External and genital differences among species are sometimes slight, so variation within the group remains poorly elucidated. The present molecular study aims to provide a clearer interpretation of the diagnostic characters and attempts to correct previous classification systems (Clarke 1955, Razowski et al. 2007). Clarke (1955) arranged the species of Orthocomotis roughly by features of male genitalia, which were the main tool for subsequent infrageneric studies (Brown 2003, Razowski et al. 2007). Orthocomotis appears to be most species rich in Brazil and Ecuador, but this may merely reflect the intensity of survey efforts in these countries. Both countries are distinct in terms of physiography, especially Ecuador, where speciation and endemism in the various ranges of the Andes is very high (e.g., Razowski & Wojtusiak 2009). The occurrence of subtle morphological differences between the species and the extent of variation of those characters within species have been little studied to date. The present paper is the first integrative morpho-molecular study of over 20 species of Orthocomotis based on specimens obtained from natural history collections.

Material and methods Specimens. The examined specimens were collected by Janusz Wojtusiak and Volker Pelz from 17 localities in Colombia, Ecuador, and Peru over the period 1998–2009 (for details see Table 1). The specimens are deposited in the collection of the Zoological Museum of the Jagiellonian University, Cracow (MZUJ) and the personal collection of Volker Pelz (VP). Among the examined specimens are several holotypes and paratypes, which is significant owing to difficulties in the identification of species that exhibit only slight morphological differences. Five species were determined to be unnamed, and these will be treated in a separate paper. Representatives of the tribe Olethreutini, i.e., Apotomis inundana, Apotomis sauciana, and Olethreutes subtilana, were used as outgroups. A list of the examined Orthocomotis species is presented in Table 1. Molecular techniques. To minimize damage to specimens, DNA was extracted from two legs (a hindleg and a midleg) of dry individuals, which had been identified based on external morphology (wing pattern) and genital characters. DNA was isolated using the NucleoSpin Tissue Kit (Macherey-Nagel, Germany). To obtain purified DNA, we applied 100 μL of Elution Buffer onto the silica membrane. To amplify a fragment of the mitochondrial COI gene, the following primer pair designed for Lepidoptera was used: LEP-F1, 5’ATTCAACCAATCATAAAGATAT-3’; and LEP-R1, 5’-TAAACTTCTGGATGTCCAAAAA-3’. If the above COI pair of primers did not yield a well-defined product, the internal primer LEP-R2 5’CTTATATTATTTATTCGTGGGAAAGC-3’ was used instead of LEP-R1. These are universal primers applied for species identification in DNA barcoding (Hebert et al. 2004). PCR amplification for all analyzed DNA fragments was conducted with a final volume of 40 μL containing 4 μL of DNA, 1.5 U Taq-Polymerase (EURx, Poland), 0.8 μL of 20 μM of each primer, 10 × PCR buffer, and 0.8 μL of 10 mM dNTPs in a Mastercycler ep (Eppendorf, Germany). The amplification protocol followed that of Hebert et al. (2004). In order to assess the quality of amplification, PCR products were electrophoresed in 1% agarose gel for 45 min at 85 V with a DNA molecular

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weight marker (Mass Ruler Low Range DNA Ladder, Fermentas, Lithuania). NucleoSpin Gel and PCR Clean-up (Macherey-Nagel, Germany) were used for purifying PCR products. In some PCR products, additional sub-bands were obtained. In such cases, 30 μL of each PCR product was separated on 1.8% agarose gel (100 V/60 min) with a DNA molecular weight marker (Mass Ruler Low Range DNA Ladder, Fermentas, Lithuania). Then the band representing the examined fragment was cut out and purified. Cycle sequencing was done in both directions with the application of BigDye Terminator v3.1 chemistry (Applied Biosystems, USA). The primers LEP-F1, LEP-R1, and LEP-R2 were used for sequencing. Each sequencing reaction was conducted with a final volume of 10 μL containing 3 μL of template, 1 μL of BigDye Master Mix (1/4 of standard reaction), 1 μL of sequencing buffer, and 1 μL of 5 μM primer. Sequencing products were precipitated using Ex Terminator (A&A Biotechnology, Poland) and separated on an ABI PRISM 377 DNA Sequencer (Applied Biosystems, USA). Sequences are available in the GenBank database (for accession numbers see Table 1). Data analysis. Sequences were examined using Chromas Lite (Technelysium, Australia) to evaluate and correct chromatograms. Alignment of sequences was performed using ClustalW (Thompson et al. 1994) within the BioEdit software (Hall 1999). Phylograms were constructed with Mega v6.0 (Tamura et al. 2013) using neighborjoining (NJ) (Saitou & Nei 1987), maximum parsimony (MP) (Nei & Kumar 2000), and maximum likelihood (ML) (Felsenstein 1981). NJ analysis was performed using the Kimura 2-parameter correction model (Kimura 1980) by bootstrapping with 1000 replicates (Felsenstein 1985). MP analysis was evaluated with the min-mini heuristic parameter (at level 2) and bootstrapping with 1000 replicates. Bayesian inference (BI) was performed with MrBayes 3.1.2 (Ronquist & Huelsenbeck 2003); the analysis was run for 5,000,000 generations, and trees were sampled every 100 generations. All trees were constructed with TreeView 1.6.6 (Page 1996). The analysis of haplotype diversity, nucleotide diversity, and variable nucleotide positions was conducted with DnaSP v5.10.01 (Librado & Rozas 2009). The analysis of nucleotide frequencies, p-distance estimation, and identification of substitution model (T92+G for COI mtDNA fragments) for ML analysis were done with Mega v6.0 (Tamura et al. 2013). The neighbor-net method (Bryant & Moulton 2004) was used to build an unrooted phylogenetic network for the studied COI gene fragments in Splitstree 4.14.2 (Huson & Bryant 2006). This method identifies and shows different or conflicting phylogenies within a single dataset by producing a tree-like network in which branch lengths correspond to well-matched collections of splits, whereas discordant splits appear as boxes.

Results and discussion Application of DNA mini-barcoding in museum collections. We sampled 120 museum specimens assigned to the genus Orthocomotis on the basis of morphological characters. In 62 cases (52%) we obtained COI sequences (Table 1). Two DNA sequences were obtained from specimens collected in the 1990s; the most recently collected individuals were from 2009 (Table 1). Because the identification of certain specimens could not be determined, the sequences were used to reveal species limits. Sequencing and analysis of this DNA fragment has been shown to be the best starting point for subsequent detailed taxonomic study (e.g., Kekkonen & Hebert 2014). In addition to the “barcode” part of the COI gene, two nuclear markers (ITS2 rDNA, and EF1a gene) were tested on the Orthocomotis species. Probably due to the poor quality of DNA isolated from the dry legs of specimens, we failed to obtain adequate sequences or even a PCR product of the above genome fragments. We were able to obtain full-length COI fragments only for 17 samples (primers pair LEP-F1-LEP-R1); however, we acquired short (307-bp) DNA fragments (primers pair LEP-F1-LEP-R2) for 45 samples. After trimming of illegible chromatogram fragments, 227-bp homologous sequences were obtained for comparison. There was no PCR product in 58 of the 120 specimens. The quality of extracted DNA is affected not only by the age of museum specimens (Watts et al. 2007), but also by the storage method (Mandrioli et al. 2006) and killing agent (Willows-Mundro & Schoeman 2015). However, different solutions have been proposed for studies of old-type specimens of high scientific value, including DNA extraction methods (Andersen & Mills 2012), the application of many primer pairs amplifying short, overlapping fragments which comprise of a complete DNA barcode sequence (Strutzenberger et al. 2012, Han et al. 2014), and the use of next generation sequencing (Prosser et al. 2016). Although analysis of full-length barcode sequences is a “gold standard” in such investigations, even 100–150-bp long (or shorter) sequences have proven useful for the identification of old specimens in a cost-effective way (Hajibabaei et al. 2006b, Meusnier et al. 2008, Mutanen et al. 2014). DNA BARCODING OF ORTHOCOMOTIS

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