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bs_bs_banner Biological Journal of the Linnean Society, 2013, 108, 68–78. With 2 figures Genetic differentiation of island populations: geographical barrier or a host switch? MAXI POLIHRONAKIS RICHMOND1, SARAH JOHNSON1, TAMARA S. HASELKORN1†, MICHELLE LAM1, LAURA K. REED2 and THERESE A. MARKOW1* 1 Division of Biological Sciences, University of California, 9500 Gilman Drive, La Jolla, San Diego, CA 92093, USA 2 University of Alabama, Department of Biological Sciences, 300 Hackberry Lane, Tuscaloosa, AL 35487, USA Received 14 May 2012; revised 26 June 2012; accepted for publication 26 June 2012 In the Sonoran desert, there exists a diverse community of cactophilic drosophilids that exploit toxic, rotting cactus tissue as a food resource. The chemistry of the necrotic cactus tissue varies among species, and several drosphilid species have evolved specialized detoxification mechanisms and a preference for certain cactus types. In the present study, we compared the genetic structure of two columnar cactus species, Drosophila mettleri and Drosophila mojavensis, and two prickly pear species, Drosophila mainlandi and Drosophila hamatofila, which have all recently colonized Catalina Island off the coast of southern California. Because there are no columnar cactus species on Catalina Island, the two columnar specialists underwent a host switch to prickly pear cactus, the only cactus present on the island. Previous genetic studies of D. mettleri and D. mojavensis showed significant genetic differentiation between mainland and island populations, which could result from restricted gene flow as a result of the San Pedro Channel, or because of a host switch to prickly pear. To distinguish between these possibilities, we analyzed the genetic structure of the prickly pear species aiming to isolate the effects of geography versus host switching. The results obtained show little to no genetic differentiation for the prickly pear species, supporting the hypothesis that the genetic differentiation of the two columnar species is a result of a host switch from columnar cacti to prickly pear. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 108, 68–78. ADDITIONAL KEYWORDS: cactophilic – California – Channel Islands – Drosophila – host specialization. INTRODUCTION Insect-host plant interactions are often associated with specific adaptations, such as specialized biochemical detoxification mechanisms, that allow insects to feed on plants with secondary metabolites that would otherwise be harmful (Krieger, Feeny & Wilkinson, 1971; Karban & Agrawal, 2002; Li, Schuler & Berenbaum, 2007; Matsuki et al., 2011). A number of Drosophila species inhabiting the arid regions of North America utilize necrotic cacti as breeding sites, and the developing larvae subse- *Corresponding author. E-mail: [email protected] †Current address: University of Rochester, Department of Biology, Rochester, NY 14627, USA. 68 quently feed on the rotting tissue and resident microfauna of yeast and bacteria, which is known to be toxic to many insect species. The chemistry profiles of the cactus tissue vary by species (more closelyrelated species have more similar chemistry) with respect to a variety of toxic compounds, such as triterpene glycosides, alkaloids, and sterols (Fogleman & Danielson, 2001). There is genetic evidence for species-specific host detoxification mechanisms in desert drosophilids and it has revealed evolutionary changes in known detoxification genes (Matzkin, 2004, 2005, 2008; Matzkin & Eanes, 2003; Matzkin et al., 2006; Bono et al., 2008). In the Sonoran and Mojave Deserts of northern Mexico, Arizona, and California, Drosophila mojavensis and Drosophila mettleri are two well-studied © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 108, 68–78 GENETIC DIFFERENTIAITION OF ISLAND POPULATIONS cactophilic drosophilid species with a broad geographic distribution. Each species is associated with a specific cactus species in different parts of its range. Drosophila mojavensis breeds in necrotic cactus tissue and D. mettleri breeds in the soil soaked with the necrotic juice surrounding the cacti (Heed, 1982, 1989). Previous studies of D. mojavensis have shown it to be a relatively strict specialist with four geographically isolated subspecies each feeding on a different cactus host: columnar cacti (Stenocereus gummosus and Stenocereus thurberi), barrel cactus (Ferocactus cylindraceus), and prickly pear (Opuntia spp.) (Heed, 1978; Heed & Mangan, 1986; Ruiz & Heed, 1988; Ruiz, Heed & Wasserman, 1990; Reed, Nyboer & Markow, 2007; Pfeiler, Castrezana & Reed, 2009). On the other hand, with the exception of the Catalina Island population, D. mettleri has little to no genetic structure throughout its mainland range, and has been found in association with columnar cactus species, Carnegiea gigantean, Pachychereus pringylei, S. thurberi, and Lophocereus schottii, as well as the California barrel cactus (F. cylindraceus) in the most northern part of its range (Heed, 1982; Markow, Castrezana & Pfeiler, 2002; Hurtado et al., 2004). Thus, these two species represent two cactophilic specialists: D. mojavensis, a relatively strict specialist, comprising geographically and genetically isolated populations that each specialize on different cactus species, and D. mettleri, a moderate specialist with a contiguous population (Bono et al., 2008). Interestingly, both D. mojavensis and D. mettleri have been collected on Santa Catalina Island, off the coast of southern California, where the only cacti available as hosts are native, and non-native, species of prickly pear (Opuntia spp.) (Millspaugh & Nuttall, 1923; Heed, 1982). This is the only portion of the range where each of these species feed on prickly pear, even though it is readily available throughout their respective distributions (Heed, 1982). Both Drosophila species thus have switched host plants on this island and both have been reported to show significant genetic differentiation from con-specific populations on the mainland (Hurtado et al., 2004; Reed et al., 2007; Haselkorn, Markow & Moran, 2009). It is unclear whether this genetic structure reflects single colonization events as a result of the inherent difficulties associated with a host switch, or if the San Pedro Channel between Catalina Island and the mainland is sufficient to reduce or prevent gene flow. One approach for evaluating the influences of host shifts and founder events on the genetics of the insular D. mojavensis and D. mettleri would be to examine additional cactophilic Drosophila species that breed in prickly pear, and are found on both sides of the channel. Several other cactophilic Drosophila species are found both on Santa Catalina Island and 69 the North American mainland. Drosophila mainlandi and Drosophila hamatofila have been reared from a number of different prickly pear species throughout their ranges (Patterson, 1943; Wasserman & Wasserman, 1992; Oliveira et al., 2005). Although D. mainlandi is restricted in its distribution to southern California and the Baja California peninsula, D. hamatofila appears to be the most widespread of the cactophilic drosophilids (Fig. 1) (Oliveira et al., 2005). In the present study, we use these four species to examine patterns of genetic differentiation associated with island colonization, and to isolate the effect of host shifts and barriers to gene flow presented by the 40-km San Pedro Channel off the coast of southern California. If the Catalina Island populations of prickly pear feeders, such as D. mainlandi and D. hamatofila, have patterns of genetic structure similar to the columnar species D. mojavensis and D. mettleri, this would suggest that the San Pedro Channel is a barrier to gene flow for these drosophilid species. On the other hand, if the Catalina Island populations of the two prickly pear species are not genetically differentiated, it will suggest the channel is not a significant barrier to gene flow, and that other factors, such as ecological adaptation required for a host switch, are contributing to the genetic isolation between island and mainland populations of D. mojavensis and D. mettleri. MATERIAL AND METHODS FOCAL SPECIES: D. MOJAVENSIS, D. METTLERI, D. MAINLANDI, AND D. HAMATOFILA Detailed phylogeographic treatments of D. mojavensis and D. mettleri have previously been published (Hurtado et al., 2004; Reed et al., 2007; Haselkorn et al., 2009). For the present study, we added data for two additional Drosophila repleta group species: D. mainlandi and D. hamatofila. All four of these cactophilic Drosophila species are found in the Sonoran Desert (Fig. 1) and have established populations on one or more of the California Channel Islands, including Catalina Island. SAMPLING AND DATA COLLECTION Taxon sampling was focused in southern California, northern Baja California, Mexico, and Organ Pipe National Monument in Arizona. All newly-collected specimens were obtained using traps baited with a banana and yeast mixture. Genomic DNA was extracted from single whole flies of field-collected specimens using DNeasy Tissue extraction kits (Qiagen), and squish preps. A portion of the cytochrome c oxidase subunit I (COI) gene was amplified using primers LCO1490f/HCO2198r in accordance © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 108, 68–78 70 M. P. RICHMOND ET AL. D. mojavensis D. mettleri D. mainlandi D. hamatofila Figure 1. Distribution map for each of the four species in the south-western USA and north-western Mexico. with standard polymerase chain reaction (PCR) conditions (Folmer et al., 1994). PCR products were purified using QIAquick PCR Purification kits (Qiagen) and ExoSAP-IT® (USB Corp.). Forward and reverse sequencing reactions and sequence visualization was performed by Genewiz, Inc., and DNA sequences were edited and aligned in SEQUENCHER, version 4.8 (GeneCodes Corp.). All gene sequences were translated to amino acids to check for stop codons, and aligned manually (no indels were present). STATISTICAL ANALYSIS The following genetic diversity indices were estimated for each species in DNAsp, version 5.10.01 (Librado & Rozas, 2009): haplotype diversity (Nei, 1987), nucleotide diversity (p) (Nei, 1987), qs per site (Watterson, 1975), and the mean number of nucleotide differences (k) (Tajima, 1996). To analyze population structure and demography, we conducted several analyses using ARLEQUIN, version 3.5.1.3 (Excoffier & Lischer, 2010). For each species, all haplotypes were grouped according to four main geographical regions (populations): Mainland (including San Diego, California, and Organ Pipe National Monument, Arizona), Peninsular (northern Baja California Peninsula, Mexico), Catalina Island, and Santa Cruz Island (D. mainlandi only). To identify limits to gene flow and population structure between the mainland and island populations, we computed pairwise FST values (Excoffier, Smouse & Quattro, 1992) between all populations. To test for significance, the data were permuted in Arlequin following the null hypothesis of no differences between populations such that the P-value represents the proportion of permutations (N = 1000) where the FST value is equal to or greater than the observed value. To determine the source of variation (within versus among populations), and estimate the covariance components (Va and Vb) for each species, we performed an analysis of molecular variance (Excoffier et al., 1992). Significance of Va (variation among populations) was tested using 1000 permutations of haplotypes among populations. Phylogenetic analysis of COI for each taxon was performed using MrBayes, version 3.1 (Huelsenbeck & Ronquist, 2001) to infer relationships among individuals. Models of molecular evolution for each © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 108, 68–78 71 GENETIC DIFFERENTIAITION OF ISLAND POPULATIONS marker were evaluated in JMODELTEST, version 0.1.1 (Posada, 2008) using the Akaike information criterion (AIC). Gene trees of all unique haplotypes were inferred for each species in MrBayes, imposing the model specified by the AIC, using default priors. We also constructed haplotype networks for each species using TCS, version 1.21 (Clement, Posada & Crandall, 2000) to obtain a nonbifurcating perspective of relationships. We used the default settings of a 95% connection limit. Because of the close relationship of all haplotypes within species, only the TCS networks are presented here because they provide more detailed information than the gene trees. RESULTS SAMPLING AND DATA COLLECTION Datasets compiled for each species included sequences from GenBank, as well as newly-sequenced specimens: 601 bp of COI for 46 D. mainlandi specimens from Mainland, Catalina Island, and Santa Cruz Island (GenBank Accession numbers JX489172– JX489217); 597 bp of COI for 32 D. hamatofila specimens from Mainland and Catalina Island (Genbank Accession numbers JX492964–JX492995); 656 bp of COI for 117 D. mettleri specimens from Mainland, Baja Peninsula, and Catalina Island (Hurtado et al., 2004) (GenBank Accession numbers AY533789– AY533812); and 570 bp of COI for 190 D. mojavensis specimens from Mainland, Baja Peninsula, and Catalina Island (Reed et al., 2007; Haselkorn et al., 2009) (GenBank Accession numbers DQ383686–DQ383730 and FJ656811–FJ656997). Note that only sequences from populations pertinent to the present study were used: CI, ANZA, COLN, SANQ, ROSO, and SARO, sensu Reed et al. (2007), and OPNM and CI, sensu Haselkorn et al. (2009). Sample sizes for all populations within each species were at 15 or greater (Table 1). Table 1. Sample sizes and genetic diversity indices Species Drosophila mainalndi (Total) Drosophila mainlandi (Mainland) Drosophila mainlandi (Catalina Island) Drosophila mainlandi (Santa Cruz Island) Drosophila hamatofila (Total) Drosophila hamatofila (Mainland) Drosophila hamatofila (Catalina Island) Drosophila mettleri (Total) Drosophila mettleri (Mainland) Drosophila mettleri (Catalina Island) Drosophila mettleri (Baja) Drosophila mojavensis (Total) Drosophila mojavensis (Mainland) Drosophila mojavensis (Catalina Island) Drosophila mojavensis (Baja) Length (bp) Polymorphic sites (parsinomy informative) Haplotypes observed Haplotype diversity p qs k 46 601 17 (5) 18 0.787 0.00218 0.00644 1.313 15 601 9 (1) 9 0.800 0.00219 0.00461 1.314 16 601 9 (2) 9 0.767 0.00283 0.00451 1.7 15 601 3 (0) 4 0.371 0.00067 0.00154 0.4 32 597 39 (18) 25 0.978 0.00977 0.01622 5.833 16 597 21 (12) 12 0.942 0.00872 0.0106 5.208 16 597 31 (10) 15 0.992 0.01052 0.01565 6.283 117 45 656 656 25 (10) 13 (5) 24 12 0.716 0.582 0.00209 0.00149 0.00714 0.00453 1.373 0.98 16 656 2 (1) 3 0.575 0.00095 0.00092 0.625 56 156 656 587 14 (4) 32 (23) 14 24 0.664 0.848 0.00141 0.00985 0.00465 0.00969 0.928 5.71 88 587 26 (21) 16 0.711 0.01031 0.00877 6.055 34 587 1 (1) 2 0.114 0.00019 0.00042 0.114 34 587 10 (2) 8 0.674 0.00255 0.00417 1.497 N p, nucleotide diversity; qs, Watterson estimator per site; k, mean number of nucleotide differences. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 108, 68–78 72 M. P. RICHMOND ET AL. Table 2. Pairwise FST values for Drosophila hamatofila Catalina Island Catalina Island Mainland 0 0.028 Table 5. Pairwise FST values for Drosophila mainlandi Mainland 0 Catalina Island Mainland Santa Cruz Island Catalina Island Mainland Santa Cruz Island 0 0.169* 0.271* 0 -0.010 0 Table 3. Pairwise FST values for Drosophila mettleri Catalina Island Catalina Island Mainland Baja Peninsula 0 0.678* 0.698* Mainland 0 0.008 Baja Peninsula 0 Significant comparisons (P < 0.05) are denoted by an asterisk (*). Table 4. Pairwise FST values for Drosophila mojavensis Catalina Island Mainland Baja Peninsula Catalina Island Mainland Baja Peninsula 0 0.506* 0.871* 0 0.228* 0 Significant comparisons (P < 0.05) are denoted by an asterisk (*). POPULATION GENETICS AND DEMOGRAPHY Of the four species included in the analysis, D. mojavensis and D. hamatofila had the highest genetic diversity (Table 1). This result was particularly interesting for D. hamatofila because this species had the smallest sample size of all four species, which means that almost every individual had a unique haplotype. Both the Mainland and Catalina Island populations of D. hamatofila had comparable genetic diversity, whereas, in D. mojavensis, the Mainland population was much more diverse than the Catalina Island population. Genetic diversity of D. mettleri and D. mainlandi was relatively low, with the Mainland and Peninsular (D. mettleri only) populations harbouring most of the genetic diversity. For all species except D. hamatofila, the Catalina Island population showed reduced gene flow with all other populations (Tables 2, 3, 4, 5). Pairwise FST values between Catalina Island and Mainland/ Peninsular populations were significant for D. mettleri, D. mojavensis, and D. mainlandi. Interestingly, the D. mainlandi population collected from Santa Cruz Island was isolated from the Catalina Island population but not from the Mainland. For two species, Significant comparisons (P < 0.05) are denoted by an asterisk (*). D. mettleri and D. mojavensis, which had samples from both Mainland and Peninsular populations, there was evidence of reduced gene flow between these populations for D. mojavensis but not for D. mettleri. Results of the analysis of molecular variance (Table 6) revealed that most of the genetic variance for D. hamatofila was based on within population variation (97.16%), with very little diversity originating from variation among populations (2.84%). A similar result was found for D. mainlandi, which harboured 82.89% of the genetic variation within populations and only 17.11% among populations. Both D. mettleri and D. mojavensis exhibited approximately equivalent levels of variation within and among populations. Haplotype networks for all species were connected using the 95% connection limit indicating a close relationship of haplotypes within species (Fig. 2). The networks for D. mettleri, D. mainlandi, and D. mojavensis were structured similarly in that they were dominated by a high frequency of a few haplotypes, with several singleton haplotypes derived from these. Dominant haplotypes for D. mettleri were shared between the Baja Peninsula and Mainland. Dominant haplotypes for D. mojavensis primarily comprised individuals from a single locality. The two distinct D. mojavensis Mainland groups comprise Drosophila mojavensis mojavensis from the Anza Borrego Desert, California, and Drosophila mojavensis sonorensis from Organ Pipe National Monument, Arizona. The dominant haplotype for D. mainlandi was found in all four localities sampled (Catalina Island, Mainland, Baja Peninsula, and Santa Cruz Island), suggesting a relatively recent colonization of Catalina and Santa Cruz Islands. The D. hamatofila network was very different from the other three in that there were no dominant haplotypes, no apparent geographical structure, and a greater number of missing haplotypes. Analyses of relationships based on the COI haplotype networks showed relatively high levels of divergence for D. mettleri and D. mojavensis, providing further support for strong, and almost complete localization and isolation of Catalina Island populations. For both these species, the haplotypes sampled © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 108, 68–78 GENETIC DIFFERENTIAITION OF ISLAND POPULATIONS 73 Table 6. Results of the analysis of molecular variance Source of variation Species Among populations within groups Drosophila Drosophila Drosophila Drosophila Within populations Drosophila Drosophila Drosophila Drosophila Sum of squares Variance components Percentage of variation hamatofila mettleri mainlandi mojavensis 4.219 27.863 4.793 195.611 0.08411 Va 0.38125* Va 0.11883* Va 1.68548* Va 2.84 45.64 17.11 47.07 hamatofila mettleri mainlandi mojavensis 86.188 51.761 24.750 326.984 2.87292 0.45404 0.57558 1.89526 97.16 54.36 82.89 52.93 Vb Vb Vb Vb Significant among population variance values (P < 0.05) are denoted by an asterisk (*). from Catalina Island were unique and not shared with any other populations. Furthermore, all D. mettleri and D. mojavensis Catalina haplotypes arose from a single ancestor, supporting a single colonization of Catalina Island. Both D. hamatofila and D. mainlandi had shared haplotypes between Catalina Island and the other populations sampled. Drosophila hamatofila had two shared haplotypes between Catalina and the Mainland, and 13 unique haplotypes on Catalina Island. For D. mainlandi, there were two shared Catalina Island haplotypes: one was the dominant haplotype of this species collected in all four populations sampled, the other was identical to a Mainland haplotype. There were seven unique D. mainlandi haplotypes on Catalina Island. Taken together, genetic analysis of the mitochondrial COI gene indicated that the two columnar species, D. mettleri and D. mojavensis, revealed more geographical structure, such that the major regions were dominated by closely-related haplotypes. On the other hand, the two prickly pear species, D. hamatofila and D. mainlandi, demonstrated less isolation among the major geographical regions, with evidence for gene flow between Catalina Island and Mainland populations. DISCUSSION Analyzing the phylogeographical structure of cactophilic drosophilids on Catalina Island provides an opportunity to test whether the 40-km San Pedro Channel is a substantial barrier to gene flow, or whether other factors determine genetic differentiation, such as evolving the necessary adaptations for a successful host switch. Based on the mitochondrial DNA sequence data reported in the present study, populations of the columnar cactus species D. mojavensis and D. mettleri showed more genetic isolation on Catalina Island than the prickly pear cactus species D. mainlandi and D. hamatofila. This result is interpreted as evidence for a role of ecological specialization in the genetic structuring of D. mojavensis and D. mettleri. ISLAND COLONIZATION The genetic data reported in the present study suggest a single colonization of Catalina Island by both D. mettleri and D. mojavensis. All of the mitochondrial haplotypes sampled from Catalina Island for both species arose from a single ancestor, and there was no evidence for gene flow between Catalina Island and either of the two mainland populations. This result parallels that of previous studies of both species based on nuclear and mitochondrial DNA sequence data (Markow et al., 2002; Hurtado et al., 2004; Reed et al., 2007). Although the origin of the D. mojavensis population that colonized Catalina appears to be from Baja, it is difficult to infer the origin of D. mettleri on Catalina Island because there is no genetic structure between the Mainland and Baja Peninsula populations (Fig. 2). As a result of evidence of gene flow between Catalina Island and the Mainland/Baja Peninsula populations for both prickly pear species, D. hamatofila and D. mainlandi, it does not appear the ocean channel operates as a genetic isolating mechanism for these drosophilids. This result is consistent with phylogeographical studies of other cactophilic drosophilid species, including D. mettleri, which support longrange dispersal (approximately 120 km) across the Sea of Cortez (Hurtado et al., 2004). Interestingly, compared with the Catalina Island data, the dispersal distance between Catalina Island and the mainland is shorter, although the genetic differentiation is higher. Because prickly pear is the only cactus host available on Catalina Island, this may be a result of increased selection pressure and decreased likelihood of a founder event. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 108, 68–78 74 M. P. RICHMOND ET AL. 2 D. mettleri D. mojavensis 61 4 14 18 45 4 3 5 3 2 12 32 7 2 9 3 2 6 3 9 Catalina Island Mainland Baja Peninsula Santa Cruz Island D. hamatofila D. mainlandi 2 3 2 4 2 20 2 8 Figure 2. TCS Haplotype networks of cytochrome c oxidase subunit I COI for each of the four species. Colours denote population origin; the size of circles is proportional to the haplotype frequency. The number of individuals with a particular haplotype, if greater than one, is provided next to the haplotype circle. Alternatively, it is possible that D. mojavensis and D. mettleri colonized the island prior to D. hamatofila and D. mainlandi, thus increasing the time available for genealogical sorting. However, if we were to calibrate the number of nucleotide substitutions in each species to a molecular clock, it is clear that D. mettleri colonized Catalina Island more recently than D. mojavensis as a result of this population being one mutational step from a mainland population, whereas D. mojavensis on Catalina Island is five mutational steps from the mainland population. We do not consider that this is an effect of sampling as a result of the large sample size of D. mojavensis on Catalina Island relative to the number of haplotypes observed. Thus, it does not appear that the results obtained in the present study reflect the amount of time since each species colonized Catalina Island. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 108, 68–78 GENETIC DIFFERENTIAITION OF ISLAND POPULATIONS GENETIC ISOLATION AND HOST SWITCHES Drosophila mojavensis and D. mettleri each made a large host switch from having their primary associations with columnar (S. gummosus, S. thurberi, and L. schotii) and barrel (F. cylindraceus) species to prickly pear (Opuntia spp.), when they moved to Catalina Island. That this is a significant host jump is supported by differences in the chemical composition and yeast communities of the different cactus species (Heed et al., 1976; Kircher, 1982; Fogleman & Danielson, 2001). Additional evidence comes from previous work quantifying host-plant specificity of cactophilic drosophilids in Sonora and Sinaloa, Mexico, which showed a discrete difference between the Drosophila species reared from columnar cactus species versus those reared from prickly pear (Ruiz & Heed, 1988). Out of 570 D. mojavensis specimens collected emerging from cactus rots of columnar and prickly pear species collected sympatrically in Playa Cochorit, Sonora, Mexico, only 2.8% were from the prickly pear species. This result demonstrates that, in this population of D. mojavensis, where both prickly pear and columnar cactus resources are available, the preferred host for D. mojavensis was the columnar cactus species. This result is particularly interesting because many columnar specialists, such as D. mojavensis and D. mettleri, were successfully reared in prickly pear in laboratory experiments (Ruiz & Heed, 1988). One suggested explanation for this success is the higher water content and elevated levels of free sugars in prickly pear species, as well as lower alkaloid levels (Kircher, 1982; Ruiz & Heed, 1988). Prickly pear and columnar species are not close relatives in the cactus phylogeny (Gibson & Horak, 1978; Cota & Wallace, 1997), and thus it is not unusual for them to have different levels of toxicity and nutrient resources (Fogleman & Danielson, 2001). In a recent study of a parallel system of cactophilic drosophilids in South America, Soto et al. (2012) suggested that host shifts of columnar specialists to prickly pear represent an easier transition as a result of lower toxicity of the prickly pear cactus tissue. Interestingly, it has been hypothesized that prickly pear species are the ancestral host for the D. mulleri complex of cactophilic drosophilids containing D. mettleri and D. mojavensis (Ruiz & Heed, 1988). Thus, a host switch for D. mojavensis and D. mettleri from columnars to prickly pear would represent a return to the ancestral condition, and may not present as many challenges as a switch to a novel host. However, host specificity in Drosophila is a result of adaptive specialization of many life-history aspects, such as olfactory cues to find hosts, oviposition preferences, and larval feeding preferences (Markow & 75 O’Grady, 2008). For example, larval feeding rates of D. mojavensis from the Sonoran Desert were significantly higher when feeding on S. thurberi (i.e. the preferred host in this region) versus Opuntia littoralis (Craft, 2010). Such specialization could explain why other columnar cactophilic specialists such as Drosophila nigrospiracula have not expanded their range into southern California. For this species, a northward range expansion into southern California would require a host switch to prickly pear or barrel cactus, which has never been observed. Thus, even if this switch is to a less toxic environment and represents a return to the ancestral condition, our results suggest that the evolution of derived adaptations may still pose a significant ecological challenge (Futuyma, Keese & Funk, 1995). This was also found at the genetic level in a study by Matzkin (2012), which showed differential expression of detoxification genes in a derived D. mojavensis population when reared on an alternate host hypothesized to be the ancestral cactus species for the D. mojavensis species cluster. On the other hand, one could easily speculate that prickly pear species such as D. mainlandi and D. hamatofila are pre-adapted to move among prickly pear host species, and thus are not restricting the colonization of Catalina Island to episodic founder events. The genetic data that we present supported this scenario and provided evidence of ongoing gene flow between the Mainland and Catalina Island populations for D. hamatofila, and a low, albeit significant, FST value for D. mainlandi. Ecological specialization of D. mojavensis on Catalina Island occurred < 0.5 Mya (Reed et al., 2007; Matzkin, 2008), and has resulted in the evolution of an island endemic that is currently recognized as one of four D. mojavensis subspecies based on morphological, genetic, and behavioural data (Pfeiler et al., 2009; Richmond, Johnson & Markow, 2012). Because this species represents a model system for studies in speciation and ecological specialization, the evolutionary history of D. mojavensis on Catalina is better understood than that of D. mettleri. In one investigation of D. mettleri from Catalina Island and mainland populations, there was some evidence for early stages of post-zygotic isolation (Markow et al., 2002). Further investigation into D. mettleri on Catalina is warranted aiming to test the hypothesis that this population is in the early stages of becoming an additional island endemic. In addition, because the genetic underpinnings of detoxification in D. mettleri have been shown to rely on P450 genes (Danielson et al., 1997, 1998), it would be particularly interesting to examine the evolutionary history of these genes in the D. mettleri populations on Catalina Island. Ongoing work investigating arthropod diversity of California’s Channel Islands has revealed that these © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 108, 68–78 76 M. P. RICHMOND ET AL. islands support levels of diversity comparable to mainland southern California, and may serve as a necessary sink for diversity in the face of developmental pressure on the mainland (Rubinoff & Powell, 2004; Chatzimanolis, Norris & Caterino, 2010; Chatzimanolis, Caterino & Richmond, unpubl. data). A recent study investigating the patterns of genetic structure of four beetle species on California’s Channel Islands revealed varying degrees of gene flow between the islands, and between the islands and the mainland (Chatzimanolis, Caterino & Richmond, unpubl. data). The genetic structure for all species was suggestive of multiple founder events, and paralleled the varying life-history traits of the beetles investigated. For example, only one beetle species studied was capable of flight, and this species had the largest number of island colonization events, in line with the results of the Drosophila species that we report in the present study. CONCLUSIONS By comparing patterns of genetic differentiation between prickly pear and columnar cactophilic drosophilid species, we were able to test how host shifts influence patterns of genetic structure. The present study indicates that restricted gene flow across the San Pedro Channel between Catalina Island and mainland southern California for two columnar cactus specialists is a result of ecological specialization. Further work testing pre- and post-zygotic reproductive isolation between mainland and Catalina Island populations of D. mettleri is necessary to determine whether this population represents an offshoot from the contiguous distribution of this species, and is in the early stages of becoming an island endemic. Additional investigation using nuclear loci would provide a means to supplement the COI data with neutral markers, as well as test candidate genes involved in the specialization process. ACKNOWLEDGEMENTS We would like to thank the Santa Catalina Island Conservancy for research and collecting permits, as well as Darcee Guttilla and Fred Starkey for their invaluable assistance with the collection. 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