Phylogeographic structure and northward range expansion in the barnacle Chthamalus fragilis

Introduction

Evaluation of population genetic discontinuities and range boundaries in coastal marine species is essential for understanding the consequences of anthropogenic stressors like climate change which may be driving range shifts, particularly poleward range expansions (e.g., Barry et al., 1995; Zacherl, Gaines & Lonhart, 2003; Dawson et al., 2010; Harley, 2011). Along the Atlantic coast of the US, Cape Hatteras and Cape Cod are especially important boundary regions (Pappalardo et al., 2014). However, because these boundaries are permeable (e.g., many species traverse the boundaries; Pappalardo et al., 2014), as are other coastal boundary regions for nearshore species (e.g., Valentine, 1966), it is necessary to evaluate each species individually. The intertidal barnacle Chthamalus fragilis is currently found along the eastern United States, extending from the Gulf of Mexico to the Atlantic coast northward up to Massachusetts (Wells, 1966; Zullo, 1963; Carlton, Newman & Pitombo, 2011), and is thought to be experiencing a northward range expansion linked to warmer temperatures (Wethey, 1984; Carlton, Newman & Pitombo, 2011). Prior to the late 19th century, C. fragilis was observed from the Chesapeake Bay area and southward. It was first observed in New England (Woods Hole, Massachusetts) in 1898, and subsequently was observed in other locations south of Cape Cod, in Buzzards Bay and Vineyard Sound (Carlton, Newman & Pitombo, 2011). More recently, it is found along the north shore of Cape Cod, from the outer Cape (Provincetown) to Sandwich at the northern end of the Cape Cod Canal (Zullo, 1963; Carlton, 2002; Wethey, 2002; Jones, Southward & Wethey, 2012). C. fragilis is a conspicuous species occupying the easily accessible upper intertidal, so it is unlikely that an earlier northern presence was overlooked, particularly as the Woods Hole region has a long history of faunal surveys.

The source of the northern C. fragilis populations is controversial. It is unknown if the barnacles dispersed via natural (e.g., ocean currents) or anthropogenic vectors (e.g., ship hull fouling), or both. C. fragilis possesses a typical biphasic life cycle, with the potential for long distance dispersal. Adults are hermaphroditic with internal fertilization and are capable of self-fertilization (Barnes & Barnes, 1958). Thus, clusters of adults are not required for reproduction as in many barnacles (Crisp, 1950). Larvae are released into the water, typically in the summer (Lang & Ackenhusen-Johns, 1981), where they pass through 6 naupliar stages and a non-feeding cyprid stage. In chthamalids, the planktonic period may last up to three weeks or more (Miller et al., 1989), allowing ample time for larval transport by ocean currents. Cyprids settle on hard intertidal substrata and metamorphose into the adult form.

C fragilis settles on artificial surfaces, and thus has a high potential for dispersal by anthropogenic transport. Sumner (1909) suggested that the relatively sudden appearance of C. fragilis in Woods Hole, MA was due to human introduction. In support of this hypothesis, Carlton, Newman & Pitombo (2011) points out that Woods Hole was home to the Pacific Guano Company between 1863 and 1889, which received potentially fouled ships from South Carolina, the type locality for C. fragilis, and elsewhere. The construction of structures such as docks, pilings, and seawalls may have provided suitable habitats along the mostly sandy shoreline south of Connecticut, also facilitating range expansion (e.g., Jones, Southward & Wethey, 2012).

The New England region has experienced warmer temperatures since the 1850s (Carlton, 2002), and warmer temperatures may have facilitated the successful dispersal and establishment of C. fragilis by releasing it from competition with the less heat-tolerant barnacle Semibalanus balanoides in the upper intertidal (Wethey, 2002). In these intertidal areas, C. fragilis is found higher, where S. balanoides, the better competitor, cannot survive (Wethey, 2002).

The goals of this study were to investigate the phylogeographic structure of C. fragilis and gain insight into the origin of northern C. fragilis populations by comparing mitochondrial cytochrome c oxidase (COI) haplotypes from several locations in Massachusetts and Rhode Island with those obtained from locations farther south, in Virginia, South Carolina, Georgia, and Florida. Thus, sampling covered a ∼2,000 km range (minimum linear separation). While confirming the source of populations that are cryptogenic (i.e., of unknown origin) can be difficult, the existence of private haplotypes shared between the northern populations and a subset of southern populations may indicate the colonization pathway (Geller, Darling & Carlton, 2010). For example, private haplotypes shared between northern and South Carolina barnacles may support the idea that barnacles arrived through transport associated with the Woods Hole guano industry (Carlton, Newman & Pitombo, 2011). Alternatively, private haplotypes shared only between northern and Chesapeake Bay—area barnacles (at the northern end of their historic range) may suggest a range expansion. We compare genetic diversity and the distribution of mitochondrial haplotypes from barnacles ranging from Massachusetts to Florida, and demonstrate significant genetic structuring between northern and southern populations. We discuss the implications of these patterns for a genetic break near Cape Hatteras and the origin of northern C. fragilis.

Materials & Methods

We collected 108 Chthamalus fragilis individuals from 11 sites along the Atlantic and Gulf coasts of North America (Table 1). We extracted genomic DNA using DNEasy Blood and Tissue and Puregene kits (Qiagen) and amplified the mitochondrial cytochrome c oxidase I (COI) gene using standard primers (Folmer et al., 1994) and protocols. We ran 25 µl PCR reactions containing 1 µl of genomic DNA in a PCR program consisting of an initial denaturation at 95°for 3 min; 35 cycles of 95° for 30 s, 48° for 30 s, and 72° for 1 min; and a final extension at 72° for 5 min. We visualized PCR products on a 1.5% agarose gel stained with GelRed (Biotium). PCR products were purified using Qiaquick PCR Purification kits (Qiagen, Hilden, Germany) and quantified using a Nanodrop 2000 spectrophotometer (Nanodrop Technologies, Wilmington, Delaware, USA). Purified products were sent to MWG Eurofins Operon for sequencing in both directions.

We assembled chromatograms and confirmed sequence quality using Geneious v. 7.1.7 (Biomatters, San Francisco, California, USA). Sequences were aligned using ClustalW (Larkin et al., 2007) with default parameters using the Geneious platform. The alignment was confirmed by eye and translated into amino acid sequences to verify that no pseudogenes were present. Sequences were deposited in GenBank (KP898760–KP898867)

Nucleotide diversity, haplotype diversity, Tajima’s D and Fu’s Fs were calculated using DnaSP (Librado & Rozas, 2009). An Analysis of Molecular Variance (AMOVA) was performed using Arlequin (Excoffier & Lischer, 2010). To examine relationships between haplotypes, we conducted a Bayesian analysis using Mr.Bayes, accessed through Geneious. The best-fit model for the Bayesian analysis was selected using the corrected Akaike Information Criterion (AICc) with jModeltest 2.1.6 (Guindon & Gascuel, 2003; Darriba et al., 2012), based on 3 substitution schemes for compatibility with Geneious. The settings in Mr. Bayes were Nst = 6, rates= invgamma, ngammacat = 4,1,100,000 generations, sampling frequency = 1,000, number of chains = 4; temperature = 0.2, and burn-in = 100.

We examined the geographic distribution of the major well-supported haplogroups recovered in the Bayesian analysis. A Mantel test was conducted using the Isolation By Distance Web Service v. 3.23 (Jensen, Bohonak & Kelley, 2005) to test for isolation by distance. Pairwise geographic distances were calculated using Google Earth following the coast with the segments connecting two shoreline points ≤20 km, reflecting plausible larval transport routes and dispersal distances. We also compared intraspecific divergences between sequences from the major haplogroups with C. proteus, a cryptic sibling species of C. fragilis (Genbank accession numbers FJ858021–FJ858040, Wares, 2001).

Results

After trimming the ends and removing 6 positions with ambiguous base calls, our alignment was 613 base pairs, with 93 unique sequences (haplotypes), and 110 polymorphic sites, of which 58 were parsimony informative. In the amino acid alignment (which included the 6 positions excluded in the nucleotide alignment), there were three amino acid substitutions: a valine for an alanine in position 6 in a Charleston, South Carolina sequence; a valine for an isoleucine in position 55 for a Woods Hole, Massachusetts sequence, and an alanine for a threonine in position 157 for a Summerland Key, Florida sequence.

For all sites, haplotype diversity was high and Tajima’s D and Fu’s Fs were negative (Table 1), which may indicate population expansion or purifying selection. However, there were no trends with latitude and none of the Tajima’s D values were significant. F_ST and AMOVA results showed significant genetic structure particularly between distant sites (Table 2), with ∼14% of the variation among populations and ∼86% of the variation within populations (Table 3). The best-fit model selected using the AICc was HKY + I + G. A Bayesian analysis conducted with this model revealed three distinct, well-supported haplogroups (i.e., clades) (Fig. 1). A neighbor-joining tree based on HKY distances also uncovered these three haplogroups (Fig. 1), and was used to assess the distinctiveness of the haplogroups with the Species Delimitation Plugin in Geneious (Rosenberg, 2007; Masters, Fan & Ross, 2011). Within each of the three haplogroups, intraclade distances were significantly smaller than interclade distances (Table 4). Rosenberg’s P_AB was 6.5E-18, 6.5E-18, 8.0E-34, for clades 1, 2, and 3, respectively (Table 4), strongly supporting reciprocal monophyly of the three haplogroups. All three haplogroups are clearly differentiated from the sister taxon Chthamalus proteus (Fig. 2).

Haplogroups differed in their geographic distribution (Table 5; Fig. 3). Haplogroup 1 was present in all New England sites and most southern sites, except Savannah and Tampa. Haplogroup 2 was well-represented in the Massachusetts and Rhode Island sites, and also present in Virginia, but not in any of the more southern sites. Haplogroup 3 was present in the Sandwich, Truro, and Woods Hole, Massachusetts sites, but not in Rhode Island. It was the most abundant haplogroup in all of the southern sites. In Savannah and Tampa, it was the only haplogroup found. The Mantel test indicated significant isolation by distance (p < 0.001).

Discussion