Patterns of DNA barcode diversity in butterfl y species (Lepidoptera) introduced to the Nearctic

379 Final formatted article © Institute of Entomology, Biology Centre, Czech Academy of Sciences, České Budějovice. An Open Access article distributed under the Creative Commons (CC-BY) license (http://creativecommons.org/licenses/by/4.0/). EUROPEAN JOURNAL OF ENTOMOLOG Y EUROPEAN JOURNAL OF ENTOMOLOGY ISSN (online): 1802-8829 http://www.eje.cz sible to explore patterns of genetic diversity and its relationship to the expansion of introduced butterfl y species. Five butterfl y species have been introduced to the Nearctic from the Palearctic. Two, Aglais io Linnaeus (Lepidoptera: Nymphalidae) and A. urticae Linnaeus (Lepidoptera: Nymphalidae), are closely related and widespread in the Palearctic. Aglais io was fi rst recorded in Montreal in 1997 (Handfi eld, 1999). The only persistent Nearctic population remains there (Nazari, 2018), but it has also been recently reported from south-central Ontario (e.g., www. inaturalist.org/observations/25521945; www.inaturalist. org/observations/41768017) and from both the east coast (e.g., www.inaturalist.org/observations/99733765; https:// www.inaturalist.org/observations/95850782; https://www. inaturalist.org/observations/97291558) and west coast (Nazari, 2018). Aglais urticae was fi rst reported from Halifax (Scott & Wright, 1972), but most subsequent records derive from New York City (Glassberg, 1992), with a sinPatterns of DNA barcode diversity in butterfl y species (Lepidoptera) introduced to the Nearctic JACOPO D’ERCOLE 1, 2 , LEONARDO DAPPORTO 3 , B. CHRISTIAN SCHMIDT 4 , VLAD DINCĂ5 , GERARD TALAVERA6 , ROGER VILA7 and PAUL D.N. HEBERT 1, 2 1 Centre for Biodiversity Genomics, Guelph, Canada; e-mails: [email protected], [email protected] 2 Department of Integrative Biology, University of Guelph, Guelph, Canada 3 ZEN lab, Department of Biology, University of Florence, Sesto Fiorentino, Italy; e-mail: leonardo.dapporto@unifi .it 4 Canadian National Collection of Insects, Arachnids and Nematodes, Agriculture and Agri-Food Canada, Ottawa, Canada; e-mail: [email protected] 5 Ecology and Genetics Research Unit, University of Oulu, Oulu, Finland; e-mail: [email protected] 6 Institut Botànic de Barcelona (IBB), CSIC-Ajuntament de Barcelona, Barcelona, Spain; e-mail: [email protected] 7 Institut de Biologia Evolutiva (CSIC-UPF), Barcelona, Spain; e-mail: roger[email protected] Key words. Introduced species, DNA barcoding, population genetics, North America Abstract. One of the main consequences of globalization is the intensifi cation of biological introductions. Because of their negative impact on environments, the early detection and monitoring of introduced species through molecular approaches is gaining increased uptake. This study assembles 2,278 DNA barcode records to examine contemporary patterns of sequence variation in mitochondrial cytochrome c oxidase I (COI) in fi ve butterfl y species introduced to the Nearctic, with a focus on Pieris rapae Linnaeus (Lepidoptera: Pieridae) and Thymelicus lineola Ochsenheimer (Lepidoptera: Hesperiidae). Parameters of genetic diversity were low (i.e., h < 0.606, π < 0.0039) for Nearctic populations of all analyzed species. Those of P. rapae and T. lineola showed marked genetic differentiation from their source populations in the Palearctic. Haplotype distributions in their Nearctic populations exposed a starburst pattern with a few common haplotypes known from Palearctic, and infrequent haplotypes diverging from them at only one or two nucleotide sites. Some uncommon haplotypes were only found in the Nearctic suggesting they originated after invasion, while others also occur in the Palearctic. This study provides an example of genetic paradox of invasion, where species often rapidly expand their distribution and become dominant in the new habitat despite their depleted levels of sequence variation. INTRODUCTION Species introductions have intensifi ed (Hulme et al., 2009) and are now one of the major contributors to biotic change (Simberloff, 2013). While most researchers oppose introductions because of their potential negative ecological impacts (Simberloff et al., 2012), the eradication of invasive species is often impossible (Davis et al., 2011). However, monitoring programs can aid their early detection and help to document their impacts on native taxa. As DNA barcoding was proposed for specimen identifi - cation (Hebert et al., 2003a), its potential for the effi cient detection of quarantine pests was soon recognized (Hebert & Gregory, 2005). Despite this, it has rarely been used to examine patterns of sequence diversity in invasive species (e.g., Valdez-Moreno et al., 2012; Porco et al., 2013). The recent development of DNA barcode reference libraries for the butterfl y faunas of North America and Europe (D’Ercole et al., 2021; Dincă et al., 2021) has made it posEur. J. Entomol. 119: 379–387, 2022 doi: 10.14411/eje.2022.039 ORIGINAL ARTICLE 380 D’Ercole et al., Eur. J. Entomol. 119: 379–387, 2022 doi: 10.14411/eje.2022.039 Genetic analysis This work examined patterns of sequence diversity in the barcode region of the mitochondrial COI gene. Indices of genetic diversity at the population level, haplotype diversity (h) (Nei, 1987), nucleotide diversity (π) (Tajima, 1983), and number of nucleotide differences (D) (Nei & Li, 1979), were estimated with Arlequin v. 3.5.1.2. (Excoffi er & Lischer, 2010). Fasta fi les were downloaded from BOLD, edited with Geneious ver. 2022.1.1, and converted to arp format with Rstudio ver. 1.3.959 for subsequent Arlequin analysis. Geographic coordinates (WGS84) were retrieved from BOLD and from iodatabase (https://github.com/leondap/iodatabase; Dapporto et al., 2022). Despite the widespread use of h and π, it is diffi cult to determine the signifi cance of their values because past studies have targeted different taxonomic groups, gene regions, and geographic ranges (Goodall-Copestake et al., 2012). The present work computed average values of haplotype and nucleotide diversity for the butterfl y species of North America (D’Ercole et al., 2021) and employed them as a point of reference between low/high values. The Exact Test (Raymond & Rousset, 1995; Goudet et al., 1996) was employed to verify the hypothesis of random distribution of individuals in their native versus introduced range. Pearson’s coeffi cient was employed to assess the correlation between h and species area, as well as h and time since introduction, with the R package ggplot2. The species area for each species was defi ned by overlaying the geographic locations of observations (https://www.inaturalist.org/observations) on a map of North America. The perimeter delimiting these locations was then drawn to estimate the area of the species distribution. Statistical approaches, ranging from specialized parametric model-based methods (e.g., Zhang et al., 2010) to more general data-driven non-parametric methods (e.g., Hsieh et al., 2016; Phillips et al., 2020), have recently been proposed to estimate various metrics of genetic diversity. Sampling completeness was assessed using the iNEXT (iNterpolation and EXTrapolation) (Hsieh et al., 2016) R package which generates accumulation curves (Chao, 1984) that can estimate the total haplotype diversity in a species. The asymptotic values of these curves were compared with the observed haplotype diversity to quantify sampling completeness (Dincă et al., 2021). TCS networks (Clement et al., 2000) were generated with PopArt ver. 1.7 (Leigh & Bryant, 2015) to depict the relationships between the Palearctic and Nearctic populations of each species. Nucleotide positions with ambiguities or missing data were masked so network construction only considered sites with full coverage. This reduced consideration to 583 base pairs (bp) for A. io, 402 bp for P. rapae, 486 bp for P. icarus, and 501 bp for T. lineola. A BLAST search (https://blast.ncbi.nlm.nih.gov/ Blast.cgi) was performed to ascertain if haplotypes detected only in the Nearctic were found outside the study area. The haplotype maps for each species were constructed using the framework described by Dapporto et al. (2022). Geographic coordinates in decimal degrees were fi rst converted to the World gle recent report from Florida (e.g., www.inaturalist.org/ observations/20129658). The broad geographic and temporal dispersion of these records suggest that both Aglais species have been introduced on multiple occasions (Zirlin, 2002). Polyommatus icarus Rottemburg (Lepidoptera: Lycaenidae), another common Palearctic butterfl y, was fi rst detected near Montreal in 2005 and has expanded its range by about 100 km in 15 years (C. Schmidt, pers. observ.). It has also been reported from Toronto and Quebec City (e.g., www.inaturalist.org/observations/65528682; www. inaturalist.org/observations/26011839), suggesting recurrent introductions. Pieris rapae Linnaeus (Lepidoptera: Pieridae) is widely distributed in the Palearctic, where it is considered a pest (Hely et al., 1982). Refl ecting its arrival in the latter half of the 19th century, this species has the longest history in the Nearctic where multiple introductions have been reported (Ryan et al., 2019). Thymelicus lineola Ochseneimer (Lepidoptera: Hesperiidae), another widespread Palearctic species, is sometimes common enough to be a pest in southern Ontario (Pengelly, 1961; Arthur, 1962). Its fi rst Nearctic specimens were collected in 1910 from London, Ontario (Saunders, 1916), but it is now widespread across the continent (Layberry et al., 1998). The present study employs DNA barcodes to characterize levels of mitochondrial COI variation in these introduced species. This work aims to document the occurrence of multiple introduction events, identify potential source regions, and to detect novel haplotypes that potentially arose after introduction. MATERIAL AND METHODS Sampling This study assembled DNA barcodes for fi ve butterfl y species introduced to the Nearctic. Overall, 2,278 barcode samples (614 from the Nearctic and 1,664 from the Palearctic) were retrieved from BOLD (boldsystems.org) and are assembled in the public dataset DS-INTRSP (dx.doi.org/10.5883/DS-INTRSP). While 60% of these sequences were included in DNA barcode libraries for European (Dincă et al., 2021; Dapporto et al., 2022) and North American (D’Ercole et al., 2021) butterfl ies, 916 new records were retrieved from BOLD. Among these samples, 101 belonged to A. io, 109 to A. urticae, 1,095 to P. rapae, 611 to P. icarus, and 362 to T. lineola (Table 1). As no Nearctic barcode records were available for A. urticae, comparison with Palearctic populations was not possible. TABLE 1. Genetic diversity indices for the fi ve species introduced to North America. N – number of sampled individuals; h – haplotype diversity (Nei, 1987); π – nucleotide diversity (Tajima, 1983); D – average number of nucleotide differences (Nei & Li, 1979); Exact Test (Raymond & Rousset, 1995; Goudet et al., 1996); ND – no available data. Species North America Europe Populations comparison NhπShared haplotypes NhπShared haplotypes DExact Test (P-values) Aglais io 3 0 0 1\1 98 0.37 0.0007 1\10 0.23 < 0.001 Aglais urticae ND ND ND ND 109 0.59 0.0045 ND ND ND Pieris rapae 432 0.51 0.0013 4\9 663 0.99 0.0038 4\47 2.49 < 0.001 Polyommatus icarus 8 0 0 1\1 603 0.93 0.0093 1\53 6.59 < 0.001 Thymelicus lineola 171 0.35 0.0005 4\6 191 0.93 0.0159 4\36 8.95 < 0.001 381 D’Ercole et al., Eur. J. Entomol. 119: 379–387, 2022 doi: 10.14411/eje.2022.039 Geodetic System (WGS84), and then transformed into Lambert azimuthal equal-area projection to obtain geographic areas of similar size. P-distances among specimens for each species were subjected to a Principal Coordinates Analysis (PCoA). The two-dimensional PCoA was plotted over a square displaying all possible color shades to display genetic distances among specimens. The most common types of color blindness were taken into consideration. Grid cells (500 km by 500 km) were employed to group nearby specimens into a single haplotype pie. The R scripts used to obtain the haplotype maps are available on the iodatabase (https://github.com/leondap/iodatabase; Dapporto et al., 2022). RESULTS Sampling Barcode records were available for Nearctic specimens for four of the introduced species (Figs 1, 2, S1, S2), but sample sizes were so low for two species (A. io – 3, P. icarus – 8) that sampling completeness was only computed for P. rapae and T. lineola (Table S1). A total of 16 and 98 unique haplotypes, contributing to 33% and 55% of the total estimated diversity, were obtained respectively for P. rapae in the Nearctic and Palearctic. This estimate suggests that 29 haplotypes await sampling in the Nearctic versus 120 in the Palearctic. Estimates of sampling completeness for T. lineola showed that 7 and 61 haplotypes were retrieved respectively from the Nearctic and Palearctic, values corresponding to 47% and 30% of their total estimated diversity. This result suggests that 8 haplotypes await discovery in the Nearctic and 144 in the Palearctic (Table S1). Genetic diversity and haplotype distribution Values for h and π were consistently low for all Nearctic populations when compared to reference values (h = 0.606, π = 0.0039). This contrasted with the high values for these metrics in Palearctic populations of P. icarus, P. rapae, and T. lineola. By contrast, A. io showed low genetic diversity in the Palearctic (Table 1). The Exact Test revealed Fig. 1. Haplotype map for 1,095 specimens of Pieris rapae (a). Colors match the bidimensional color space of the PCoA projection (b). Fig. 2. Haplotype map for 362 specimens of Thymelicus lineola (a). Colors match the bidimensional color space of the PCoA projection (b). 382 D’Ercole et al., Eur. J. Entomol. 119: 379–387, 2022 doi: 10.14411/eje.2022.039 a non-random distribution of haplotypes in the Nearctic and the Palearctic populations for each of the four species (p-value < 0.001) (Table 1). Pearson’s coeffi cient revealed a moderately strong but non-signifi cant positive correlation between h and geographic range (R = 0.87; p-value = 0.13) (Fig. S3) and a signifi cant correlation between h and time since introduction (P = 1; p-value = 0.0012) (Fig. S4). However, because these correlations are only based on four data points, results need to be interpreted with caution. The sole Nearctic haplotypes for A. io and P. icarus were, as expected, also dominant in the Palearctic (Figs S1, S2). Among the ten haplotypes detected in Nearctic P. rapae, the dominant one was also abundant in the Palearctic. The other Nearctic haplotypes showed just one or two nucleotide substitutions from those in the Palearctic, but fi ve were only detected in the Nearctic (Fig. 3). Two of the seven Nearctic haplotypes of T. lineola were common and known from the Palearctic. The other fi ve haplotypes were infrequent, and three of them were only found only in the Nearctic (Fig. 4). A BLAST search for the haplotypes detected only in the Nearctic confi rmed they were exclusive to this continent. DISCUSSION The capacity of DNA barcoding to both assign specimens to a species (Hebert et al., 2003b) and to reveal intraspecifi c variation (e.g., Hebert et al., 2004; Burns et al., 2007; Dapporto et al., 2022) motivated its present use to compare patterns of genetic diversity in populations of butterfl y species introduced to the Nearctic with those in their ancestral Palearctic populations. While a few barcode records can provide an overview of intraspecifi c diversity, many more specimens need to be analyzed to obtain a detailed understanding of large-scale patterns of intraspecifi c variation (Zhang et al., 2010; Phillips et al., 2019). Although sequences for 432 Nearctic specimens were available for P. rapae, analysis indicated that they only represented 33% of its estimated haplotype diversity. Fewer samples (171) were available for T. lineola, but they represented 47% of its estimated haplotype diversity. The limited distribution and coverage for the A. io and P. icarus prevented their detailed analysis. However, a single haplotype was detected in the Nearctic for both, and these haplotypes were prevalent in the Palearctic as expected for recent introductions. DNA barcodes for A. urticae were unavailable in the Nearctic. Introductions often involve a population bottleneck that results in reduced genetic diversity (Baker & Stebbins, 1965). Although theory suggests such populations are at enhanced risk of extinction (Gilpin & Soulé, 1986; Lacy, 1987; Frankham, 1998), many introduced species thrive — a phenomenon termed the “genetic paradox of invasion” Fig. 3. Haplotype network for 1,095 barcoded specimens of Pieris rapae. The number of hypothesized mutational steps is indicated by hatch marks; circle size is proportional to the number of samples. Fig. 4. Haplotype network for 364 barcoded specimens of Thymelicus lineola. The number of hypothesized mutational steps is indicated by hatch marks; circle size is proportional to the number of samples. 383 D’Ercole et al., Eur. J. Entomol. 119: 379–387, 2022 doi: 10.14411/eje.2022.039 (Allendorf & Lundquist, 2003). Nearctic populations of P. rapae and T. lineola follow this pattern as they are common and widely distributed despite their low genetic diversity. Specifi c ecological features can however outweigh the negative effect of decreased genetic diversity (Estoup et al., 2016). These two species are generalists that feed on diverse plants within a family (Brassicaceae and Poaceae, respectively), tolerate varied climatic regimes (Tolman & Lewington, 2008), and are also adapted to anthropic habitats (Hufbauer et al., 2012). Larvae of P. rapae feed on agricultural crops (Brassica spp.) as well as many brassicaceous weeds common in anthropic landscapes (Lafranchis, 2007; Tolman & Lewington, 2008). Similarly, T. lineola favors timothy grass (Phleum pratense) as a larval host plant (Lafranchis, 2007; Tolman & Lewington, 2008). Polyommatus icarus has also spread in disturbed landscapes, gaining a foothold via the European host plant Lotus corniculatus (Rivest & Kharouba, 2021). Introduced populations can also experience reduced purifying selection because they often host fewer parasitoids and have fewer predators than populations in their native range (Torchin et al., 2003; Perkins et al., 2008). As a result, they can invest more in growth rate and dispersal (Muller-Scharer et al., 2004). It is worth emphasizing that conclusions derived from a single gene marker describe only part of the evolutionary history of an organism. As a result, the reduced diversity observed at mtDNA might not mirror diversity at nuclear loci (PetitMarty et al., 2021). Not only is mtDNA more exposed to population bottlenecks because of lower number of gene copies, but it is also prone to selective sweeps because of its near absence of recombination (Leffl er et al., 2012). It is therefore possible that adaptive potential is maintained, despite the reduced diversity observed at mtDNA. In agreement with earlier work on P. rapae (Scudder, 1886; Andow et al., 1990; Ryan et al., 2019) and T. lineola (Burns, 1966), the present results suggest multiple introductions. Because each introduction likely involved just a few closely related individuals from a single Palearctic locality, the presence of multiple widespread haplotypes shared with populations in the native range supports recurrent introductions. Aside from a few common haplotypes, Nearctic populations of P. rapae and T. lineola possessed infrequent haplotypes one or two mutations away from the dominant lineages. This starburst pattern is the rule in animal lineages (e.g., Chen et al., 2004; Bollongino et al., 2006; Lait & Hebert, 2018) including butterfl ies (Dincă et al., 2021), but the detection of haplotypes private to the Nearctic is surprising. While their presence in the Palearctic may have been overlooked due to incomplete sampling and introduction from other regions of the world cannot be excluded, they may also refl ect variants that arose after introduction. Based on standard rates of molecular evolution of 1.5% per million years (Quek et al., 2004), de novo variants would not be expected given the brief interval since introduction. However, because of the reduced impact of selection and drift at the population level over short time scales, mutation rates for mitochondrial DNA can vary 100-fold from the slow phylogenetic rate to the highest pedigree-based rate (Ho et al., 2005). While this shift might be responsible for the newly arisen substitutions, other factors including increased positive selection (Gillespie, 2001), relaxed purifying selection (Henn, 2009), and gene surfi ng associated with population expansion (Excoffi er & Ray, 2008) can aid the fi xation of new variants. Despite low genetic diversity in their Nearctic populations, positive linear relationships were detected between the time since introduction and geographic range. In agreement with the preceding considerations, it is possible that a combination of multiple introduction events and newly arisen mutations has led to increased diversity through time. Because the geographic area occupied is expected to co-vary with time for populations increasing in size, the observed correlation between genetic diversity and area is not surprising. Moreover, the occupancy of larger areas can, in itself, promote adaptive ecological changes and increase diversity. While this association is well-established for species diversity, the impact of area on genetic diversity is less clear (Dapporto et al., 2019; Lawrence et al., 2020). Identifying source populations in their native range and admixture of different lineages in the novel environments is critical to determine the capacity to disperse and the evolutionary potential of introduced species (Colautti & Lau, 2015; Dlugosh et al., 2015). Such information is relevant because admixture following multiple introductions from different source regions produces both an increase in genetic diversity and a progressive increase in fi tness as a result of dominance (Davenport, 1908) and over-dominance (East, 1908; Shull, 1908). Because this study revealed no clear spatial genetic structure for P. rapae in the Palearctic, it was not possible to identify the source region for the Nearctic populations. The same pattern was observed in a study that examined nuclear genomic diversity (Ryan et al., 2019). The evidence for the lack of structure is likely explained by the high dispersal capacity (Dapporto et al., 2019) and human-mediated transport (Jones et al., 1980). By comparison, Thymelicus lineola displayed three geographically distinct haplogroups in the Palearctic (Dapporto et al., 2022), and only one was found in the Nearctic. This likely excludes introductions from the Iberian Peninsula, eastern Europe, and Asia. As expected for recent introductions, a single haplotype widespread in the native range was detected in the Nearctic for A. io and P. icarus. While it is diffi cult to identify potential source regions for A. io due to the lack of clear genetic structure in its native range, P. icarus is highly structured in Europe (Dincă et al., 2011; Arif et al., 2021) and the most probable origin for the North American population is anywhere but the Iberian Peninsula. Continuous monitoring of these species will be valuable as the evolution of invading populations is rapid and unpredictable immediately after introduction. Not only a lag time often exists between the initial colonization and the onset of population expansion (Sakai et al., 2001; Bock et al., 2015), but abrupt changes in population size can also occur as a result of varying selective pressure shortly after colonization (Phillips et al., 2010). 384 D’Ercole et al., Eur. J. Entomol. 119: 379–387, 2022 doi: 10.14411/eje.2022.039 This study has compared patterns of genetic diversity in four butterfl y species introduced to the Nearctic with those of their Palearctic ancestral populations. The two most recently introduced species, A. io and P. icarus possess a single haplotype widespread in Europe. While levels of genetic diversity were much lower in Nearctic than Palearctic populations of P. rapae and T. lineola, both have broad distributions. They also displayed a starburst haplotype network in the Nearctic with a few widespread haplotypes and infrequent ones with one or two mutations. Some rare haplotypes were only found in the Nearctic, suggesting the possibility of their origin after introduction. Supporting conclusions based on their disjunct distributional patterns in the Nearctic, the analysis of haplotype variation for P. rapae and T. lineola suggest multiple introductions. Analysis of sequence variation in the native range also identifi es potential source regions for T. lineola and P. icarus. Overall, this study demonstrates the effectiveness of DNA barcoding for describing the diversity of introduced species and establish its value to monitoring programs of invasive species. ACKNOWLEDGEMENTS. We thank D. Szaboova, A. Johnsen, A. Hausmann, J. Reil, M. Seidensticker, J. Kjaerandsen, A. Segerer, V. Lukhtanov, M. Franzen for granting use of their DNA barcode records. FUNDING. This study was supported, in part, by a NSERC Discovery grant to PDNH and by an award from the Canada First Research Excellence Fund for the support of Food from Thought research program. This research was also supported by the Academy of Finland (Academy Research Fellow, decision no. 328895) to VD, by project PID2020-117739GA-I00/MCIN/AEI/ 10.13039/501100011033 to GT, by project PID2019-107078GBI00/MCIN/AEI/ 10.13039/501100011033 to RV, and by projects related to the Direttiva Biodiversità del Ministero della Transizione Ecológica to LD. COMPETING INTERESTS. The authors declare no competing interest. REFERENCES ALLENDORF F.W. & LUNDQUIST L.L. 2003: Introduction: population biology, evolution, and control of invasive species. — Conserv. Biol. 17: 24–30. ANDOW D.A., KAREIVA P.M., LEVIN S.A. & OKUBO A. 1990: Spread of invading organisms. — Landsc. Ecol. 4: 177–188. 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Species Locality N H R T Pieris rapae North America 432 16 0.33 45 Europe 663 98 0.55 178 Thymelicus lineola North America 171 7 0.47 15 Europe 191 61 0.30 205 Fig. S1. Haplotype map for 101 specimens of Aglais io (a). Colors match the bidimensional color space of the PCoA projection of pdistances (b). 387 D’Ercole et al., Eur. J. Entomol. 119: 379–387, 2022 doi: 10.14411/eje.2022.039 Fig. S2. Haplotype map for 611 specimens of Polyommatus icarus (a). Colors match the bidimensional color space of the PCoA projection of p-distances (b). Fig. S3. Pearson’s correlation between haplotype diversity (h) and area (km2); the shaded area depicts 95% confi dence interval. Fig. S4. Pearson’s correlation between haplotype diversity (h) and time since introduction (years); the shaded area depicts 95% confi dence interval.