Mitochondrial Phylogeny of Central and North American Chlosyne Lacinia (Lepidoptera: Nymphalidae)
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Eur. J. Entomol. 108: 529–535, 2011 http://www.eje.cz/scripts/viewabstract.php?abstract=1649 ISSN 1210-5759 (print), 1802-8829 (online) One variable species or multiple cryptic? Mitochondrial phylogeny of Central and North American Chlosyne lacinia (Lepidoptera: Nymphalidae) TIMOTHY C. BONEBRAKE1*, WARD B. WATT 1, ALEJANDRO PEREZ1 and CAROL L. BOGGS1 1Department of Biology, Stanford University, Stanford CA 94305-5020, USA Key words. Phylogeny, genetics, tropical, cryptic species, Melitaeini, Chlosyne, El Salvador Abstract. Recent efforts to catalogue global biodiversity using genetic techniques have uncovered a number of “cryptic” species within morphologically similar populations that had previously been identified as single species. Chlosyne lacinia (Lepidoptera: Nymphalidae), with a range extending from the Southwest U.S. to South America, is one of the most phenotypically variable and broadly distributed butterfly species in the New World. We sampled populations of C. lacinia in two temperate locations (California and Arizona) and one tropical location (El Salvador) to determine if cryptic species were present at this scale (temperate vs. tropical). We examined mtDNA sequence variation in COI, COII, the intervening tRNA (Leucine-2), 16S, 12S and an additional intervening tRNA (Valine), accounting for approximately 20% of the mitochondrial genome (3479 bp). Among all C. lacinia indi- viduals, sequence divergence did not exceed 0.0084 compared to a 0.06 estimated divergence between C. lacinia and congener C. leanira. We also found subclade structure which did not clearly correspond to geography or subspecific designation. Though the mitochondrial phylogeny suggests a complex evolutionary history and biogeography, we demonstrate that one C. lacinia species is distributed throughout North and Central America spanning a diverse set of temperate and tropical habitats. INTRODUCTION Chlosyne lacinia (Geyer, 1837) is one of the most vari- Cryptic species, morphologically indistinguishable or able and widespread butterflies in the Americas (Higgins, highly similar entities found to be distinct species, have 1960) and a member of the well-studied Melitaeini but- become a recent focus of much ecological and evolu- terfly tribe (Harvey, 1991; Ehrlich & Hanski, 2004; Nym- tionary study since the advent of PCR and molecular eco- phalidae Systematics Group, 2009). While Chlosyne is logical approaches (Bickford et al., 2007). The presence represented by species with both Nearctic and Neo- and lack of awareness of cryptic species leads to the tropical distributions, Wahlberg & Zimmerman (2000) potential to vastly underestimate biological diversity present phylogenetic evidence based on mtDNA sequence (Hebert et al., 2004; Smith, 2006; Burns et al., 2008; data to suggest that C. lacinia has colonized Central and Lohman et al., 2010), while also presenting complications South America relatively recently and that its sister spe- for the management of rare and endangered taxa (Schön- cies are Nearctic in distribution. Could it be that this Neo- rogge et al., 2002; Griffiths et al., 2010). tropcial colonization has led to the rapid radiation of mul- The distribution of cryptic species is especially impor- tiple cryptic species? Or alternatively, has one species tant across large geographic scales. Species thought to be successfully colonized multiple habitats across large cli- common and widely distributed are instead frequently matic and habitat gradients? found to be multiple cryptic species across a range (Jones We examined mitochondrial sequence variation in C. & van Parijs 1993; Colborn et al., 2001; Hebert et al., lacinia among the genes COI, COII, 12S rDNA, 16S 2004; Fernandez et al., 2006; Stuart et al., 2006; Murray rDNA, and two tRNAs following the approach of Wheat et al., 2008; Wheat & Watt 2008; Belyaeva & Taylor, & Watt (2008). In total we sequenced 3,479 bp repre- 2009; Geurgas & Rodrigues, 2010; McLeod, 2010). Even senting about 20% of the mitochondrial genome in 14 C. when cryptic species are revealed, their own particular lacinia butterflies from one tropical population (El Salva- distributions are important to consider. Multiple sym- dor: n = 6) and two temperate populations (California: n = patric cryptic species throughout a distribution differs 3, Arizona: n = 5). We focused on mitochondrial DNA greatly in terms of conservation management from allo- due to its effectiveness in marking species boundaries in patric cryptic species with largely disjunct distributions butterfly systematics (Sperling, 2003). Specifically we (i) (McBride et al., 2009). However, species exhibiting examined sequence divergence across all C. lacinia indi- extensive distributions do not necessarily have cryptic viduals and (ii) employed Bayesian and maximum likeli- associations. For example, Lohman et al. (2008) showed hood phylogenetic methods to determine whether or not that Lampides boeticus (Lepidoptera), a butterfly distrib- any distinct mitochondrial lineages were present, in par- uted across four continents, did not have any clear cryptic ticular in relation to geography (tropical vs. temperate). species complexes across its range. We used these data to address the question of whether the * Current address: Department of Atmospheric and Oceanic Sciences, UCLA, Los Angeles CA 90095-1565, USA; e-mail: [email protected] 529 TABLE 1. Locations and IDs of specimens used in this study. Details for Colias meadii and Hipparchia autonoe sequences and specimens can be found in Wheat & Watt (2008) and Kim et al. (2010) respectively. Details on NW32-2, NW14-4, JM6-8, and JM7-6 can be found at the Nymphalidae Systematics Group (2009) voucher specimen database. Accession numbers for Chlosyne listed in the table are for COI. Additional sequences are also available through Genbank (Accession nos JN584555–JN584629). County/ Collection Genbank Species ID Country/State Location Latitude Longitude Department Date Accession # EU583920 Colias meadii – U.S./Colorado – Baldy Chato 38N 107W – EU583866 Hipparchia – Korea – Halla Mt. 33N 126E May 2008 GQ868707 autonoe Chlosyne leanira – U.S./Arizona Cochise SWRS 31.9N 109.2W Aug. 2007 JN584537 Chlosyne lacinia 68 U.S./Arizona Cochise Highway 80 31.6N 109.1W 21 Aug. 2008 JN584550 72 U.S./Arizona Cochise Highway 181 31.9N 109.5W 26 Aug. 2008 JN584551 152 U.S./Arizona Cochise Paradise Cemetery 31.9N 109.2W 7 Aug. 2008 JN584548 202 U.S./Arizona Cochise Highway 181 31.9N 109.5W 26 Aug. 2008 JN584556 263 U.S./Arizona Cochise Paradise Cemetery 31.9N 109.2W 7 Aug. 2008 JN584547 NW32-2 U.S./Arizona Cochise Huachuca Mts. 31.5N 110.3W 20 May 1998 AF187779 42 U.S./California Riverside Indio – 58th St. 33.6N 116.2W 23 July 2007 JN584540 128 U.S./California Riverside Indio – 52nd St. 33.7N 116.2W 23 July 2007 JN584542 241 U.S./California Riverside Indio – 58th St. 33.6N 116.2W 23 July 2007 JN584546 NW14-2 U.S./Texas – South Texas – – 17 Feb. 1999 JN584554 59 El Salvador Ahuachapán Serrano Property 13.8N 90.0W 13 Dec. 2008 JN584543 198 El Salvador Ahuachapán Serrano Property 13.8N 90.0W 3 Dec. 2008 JN584549 210 El Salvador Ahuachapán Serrano Property 13.8N 90.0W 17 Nov. 2008 JN584545 224 El Salvador Ahuachapán Serrano Property 13.8N 90.0W 8 Nov. 2008 JN584539 255 El Salvador Santa Ana Lake Coatepeque 13.9N 89.5W 11 Dec. 2008 JN584544 258 El Salvador Ahuachapán Serrano Property 13.8N 90.0W 28 Nov. 2008 JN584541 JM6-8 Mexico/Morelos Yautepec CEPROBI 18.5N 99.1W 28 Aug. 2007 JN584552 JM7-6 Mexico/Morelos Yautepec CEPROBI 18.5N 99.1W 9 Aug. 2007 JN584553 extensive distribution of C. lacinia is the result of one 72 and 202) were collected from the field as larvae and raised to highly variable butterfly species or if instead it is a com- adults before being used in molecular data collection. For out- plex of multiple cryptic species. groups, we used a Chlosyne leanira adult collected from the Southwestern Research Station (SWRS) in Arizona, and extant MATERIAL AND METHODS mitochondrial sequence data from Hipparchia autonoe (Nym- phalidae) (Kim et al., 2010) and Colias meadii (Pieridae) Study organism and sites (Wheat & Watt, 2008) as additional outgroups. We also used Chlosyne lacinia is a morphologically highly variable but- previously sequenced COI C. lacinia samples available from the terfly with populations distributed from the southern U.S. to Nymphalidae Systematics Group (2009). northern Argentina (Higgins, 1960; Scott, 1986). Larvae use a We sliced the posterior 3–4 abdominal segments of each but- variety of host plants within Asteraceae: Heliantheae, and habi- terfly and incubated them overnight in a solution of 180 µl tats range from desert washes to open tropical secondary growth phosphate-buffered saline solution (Gibco) and 20 µl of Prote- areas (Neck, 1973; DeVries, 1987; Stewart et al., 2001). While inase K at 55°C to improve DNA yield from the dried speci- the habits and life histories of temperate C. lacinia have been mens. We then extracted DNA using a DNeasy kit (Qiagen) studied in some detail, especially in Texas (e.g. Neck, 1973), following standard protocol. We focused on sequences from much less is known about the ecology of tropical C. lacinia COI, COII, the intervening tRNA (Leucine-2), 16S, 12S and an (DeVries, 1987). Higgins (1960) cautiously categorized C. lac- additional intervening tRNA (Valine). For amplification and inia forms into several types or “subspecies” noting that the bio- sequencing of these genes, we used existing primers (Caterino logical reality behind the types would be difficult to justify & Sperling, 1999; Chew & Watt, 2006; Wheat & Watt, 2008) given complete gradation among all types and that even single with some additional C. lacinia-specific primers for amplifica- broods are capable of producing multiple types (Edwards, tion and sequencing in the case of COII (Table 2). For amplifi- 1893). This has led some (e.g. DeVries, 1987) to ignore com- cation we performed PCR using Hi-Fi Platinum Taq in a pletely the subspecific designations. Stratagene Robocycler using cycling conditions of 1 cycle of We collected Chlosyne lacinia adults from populations in 3´30˝ 94°C melt, 1´30˝ 52°C anneal, 3´30˝ 68°C extend; 35 California, Arizona and El Salvador (Table 1).