Concerted Genetic, Morphological and Ecological Diversification In

Molecular Ecology (2011) 20, 1936–1951 doi: 10.1111/j.1365-294X.2011.05065.x

Concerted genetic, morphological and ecological diversiﬁcation in Nacella limpets in the Magellanic Province

C. A. GONZA´ LEZ-WEVAR,* T. NAKANO,† J. I. CAN˜ ETE‡ and E. POULIN* *Instituto de Ecologı´a y Biodiversidad, Departamento de Ciencias Ecolo´gicas, Facultad de Ciencias, Universidad de Chile, Las Palmeras # 3425, N˜ un˜oa, Santiago, Chile, †Department of Geology and Paleontology, National Museum of Nature and Science, Tokyo, Japan, ‡Departamento de Recursos Naturales, Universidad de Magallanes, Punta Arenas, Chile

Abstract Common inhabitants of Antarctic and Subantarctic rocky shores, the limpet genus Nacella, includes 15 nominal species distributed in different provinces of the Southern Ocean. The Magellanic Province represents the area with the highest diversity of the genus. Phylogenetic reconstructions showed an absence of reciprocal monophyly and high levels of genetic identity among nominal species in this Province and therefore imply a recent diversification in southern South America. Because most of these taxa coexist along their distribution range with clear differences in their habitat preferences, Nacella is a suitable model to explore diversification mechanisms in an area highly affected by recurrent Pleistocene continental ice cap advances and retreats. Here, we present genetic and morphological comparisons among sympatric Magellanic nominal species of Nacella. We amplified a fragment of the COI gene for 208 individuals belonging to seven sympatric nominal species and performed geometric morphometric analyses of their shells. We detected a complete congruence between genetic and morphological results, leading us to suggest four groups of Nacella among seven analysed nominal species. Congruently, each of these groups was related to different habitat preferences such as bathymetric range and substrate type. A plausible explanation for these results includes an ecologically based allopatric speciation process in Nacella. Major climatic changes during the Plio-Pleistocene glacial cycles may have enhanced differentiation processes. Finally, our results indicate that the systematics of the group requires a deep revision to re-evaluate the taxonomy of Nacella and to further understand the Pleistocene legacy of the glacial cycles in the southern tip of South America.

Keywords: adaptive radiation, ecological speciation, Nacella, Patagonia, Patellogastropoda, Southern Ocean Received 5 November 2010; revision received 27 January 2011; accepted 9 February 2011

2004, 2007). Typically, taxonomic studies in the group Introduction have been based on shell morphology and external True limpets of the order Patellogastropoda are the characters, but the high degree of variability and homo- basal group in the evolution of Gastropoda, as revealed plasy detected in form and coloration has led to taxo- by morphological and molecular studies (Ponder & nomic confusion (Ridgway et al., 1998; Sasaki 1999; Lindberg 1997; Lindberg 1998; Harasewych & McArthur Espoz et al. 2004; Nakano & Spencer 2007; Lindberg 2000; McArthur & Harasewych 2003; Nakano & Ozawa 2008). Recent molecular studies have greatly improved the systematics and taxonomy of Patellogastropoda at the levels of family, genus and species. Seven families, Correspondence: C. A. Gonza´lez-Wevar, Fax: 056 02 2727363; Lotiidae, Acmaeidae, Pectinodontidae, Patellidae, E-mail: [email protected] Lepetidae, Eoacmaeidae and Nacellidae, are currently

2011 Blackwell Publishing Ltd CONCERTED EVOLUTION IN MAGELLANIC LIMPETS 1937 recognized in the order using different molecular ward through the west wind drift. However, this markers (Nakano & Ozawa 2007). hypothesis has been rejected by recent phylogenetic Nacellidae includes two genera, Cellana and Nacella, analyses that showed that the Magellanic species of with clearly disjoint distributions (Powell 1973; Lind- Nacella are the most derived clade in the genus (Gonza´lez- berg 1998, 2008). Cellana has more than 37 species and Wevar et al. 2010a). Based on morphological studies, at subspecies mainly distributed in tropical to warm- least seven species of Nacella have been described for temperate waters of the Indo-Pacific regions (Powell the Magellan Province, but the taxonomy of the genus 1973; Lindberg 1998). Its distribution range includes in this region is still unclear (Table 1). Powell (1973) southern South Africa, the east coast of Africa, Egypt, recognized five valid taxonomic units: N. deaurata, the Arabian Sea and expands eastward as far north as N. flammea, N. fuegiensis, N. magellanica and N. mytilina, Japan. Recently, one species of Cellana was reported in one ecomorph (N. deaurata form delicatissima) and the Atlantic coast of Africa, but it appears to have been two subspecies (N. magellanica venosa and N. magella- introduced from Indo-Pacific regions (Nakano & Espin- nica chiloensis). Recently, Valdovinos & Ru¨ th (2005) in a osa 2010). Cellana is also distributed in oceanic islands revision of Nacella from southern South America con- of the Indian and Pacific Oceans such as the Bonin cluded that all the morphological species described in Islands, Guam, the Hawaiian islands, Society Island the Magellanic Province, with the exception of N. fuegi- and the Juan Fernandez archipelago in the East Pacific. ensis (considered as synonym of N. magellanica by the Finally, its southernmost distribution reaches Australia authors), are true taxonomic units with diagnostic dif- and New Zealand, as far south as subantarctic islands ferences in shell morphology (thickness and coloration), such as Auckland, Snares, Bounty, Antipodes, Chatham, mantle tentacles, foot coloration and radular tooth mor- and Campbell. In the Campbell islands, Cellana strigilis phology. strigilis co-exists with its sister genus Nacella, specifically The Magellanic species of Nacella also display ecologi- N. terroris (Powell 1973). The genus Nacella comprises cal, bathymetrical and latitudinal distribution differ- 15 nominal species mainly distributed in four of the ences (Morriconi & Calvo 1993; Morriconi 1999; Rıós described biogeographical provinces of the Southern et al. 2003; Bazterrica et al. 2007). The species N. magel- Ocean (Griffiths et al. 2009): Antarctica, the Magellanic lanica, N. deaurata, N. delicatissima and N. fuegiensis are Province and subantarctic islands of the Antipodean commonly found in intertidal rocky environments, and Kerguelenian provinces (Powell 1973). One South while N. flammea and N. mytilina are subtidal (Fig. 1a). American species, Nacella clypeater, expands its distribu- Most of the species live attached to rocky substrates tion north up to Arica (Peruvian) along the Humboldt where they graze on microalgae, diatoms and bacterial Current System (Valdovinos & Ru¨ th 2005). films. Only N. mytilina lives exclusively attached to kelp In the Magellanic Province, Nacella represents one of such as Macrocystis pyrifera (Valdovinos & Ru¨ th 2005) the dominant groups of marine benthic macroinverte- and Lessonia flavicans (C.A. Gonza´lez-Wevar, personal brates, especially on rocky boulders and rocky shores observation). In terms of geographical distribution, all along the Magellan Strait and Patagonia (Guzmań N. delicatissima, N. magellanica and N. deaurata have a 1978; Rıós et al. 2003; Bazterrica et al. 2007). According wide distribution, from Chiloe´ Island in the Pacific to to Powell (1973), and considering that more than 50% the Buenos Aires in the Atlantic, including the Magellan of the species have been described from this Province, Strait, Tierra del Fuego, Southern Patagonia and the it has been considered as the centre of origin and diver- Falkland Islands (Powell 1973; Valdovinos & Ru¨th 2005). sification of the genus, from whence it expanded east- Other species such as N. flammea and N. fuegiensis exhibit

Table 1 Taxonomy of Nacella in the Magellanic Province according to four different studies. Filled squares (grey) indicate how the particular study supports synonymies between and among taxa

Nominal species Powell (1973) Valdovinos & Ru¨ th (2005) de Aranzamendi et al. (2009) This study

N. fuegiensis N. fuegiensis N. magellanica n.i. N. deaurata N. deaurata N. deaurata N. deaurata N. deaurata N. delicatissima N. delicatissima Ecotype of N. magellanica or N. deaurata n.i. N. magellanica N. magellanica N. magellanica N. magellanica N. magellanica N. chiloensis N. chiloensis n.i. N. venosa N. venosa n.i. N. mytilina N. mytilina N. mytilina N. mytilina N. mytilina N. flammea N. flammea N. flammea n.i. N. flammea n.i., not included in the analyses.

2011 Blackwell Publishing Ltd 1938 C. A. GONZA´ LEZ-WEVAR ET AL.

(a) Nacella magellanica

2 m Nacella deaurata 1 m

–1 m

Nacella Nacella mytilina fuegiensis –5 m

–10 m

Nacella flammea

–25 m

(b) –80º –75º –70º –65º –60º –55º –80º –75º –70º –65º –60º –55º –80º –75º –70º –65º –60º –55º –30º –30º –30º –30º (i) (ii) (iii)

–35º –35º –35º –35º

–40º –40º –40º –40º

–45º –45º –45º –45º

–50º –50º –50º –50º

–55º –55º –55º –55º

–80º –75º –70º –65º –60º –55º –80º –75º –70º –65º –60º –55º –80º –75º –70º –65º –60º –55º

Fig. 1 (a) Bathymetrical distributions of the nominal species of Nacella in Punta Santa Ana, Magellan Strait. (b) Latitudinal distribution of the analysed nominal species of Nacella along the Magellan Province based on the descriptions of Powell (1973) and Valdovi- nos & Ru¨ th (2005). (i) N. magellanica, N. deaurata; (ii) N. ﬂammea, N. fuegiensis and N. mytilina; (iii) N. chiloensis and N. venosa. a narrower distribution, from Ayse´n (4532¢LS; supporting the monophyly of both Nacella and Cellana 7204¢LW) to the Magellan Strait (Valdovinos & Ru¨ th and their sister relationship (Gonza´lez-Wevar et al. 2005). N. chiloensis and N. venosa are restricted to Chiloe 2010a). High levels of genetic divergence among Nacella Island (Powell 1973). Finally, N. mytilina has the widest lineages belonging to different Southern Ocean prov- distribution; it is found in the Magellan Strait, Cape inces support the existence of transoceanic discontinu- Horn, southern Patagonia, the Falkland Islands and also ities in the genus along its distribution. These genetic Kerguelen Island in the Kerguelenian Province (Powell discontinuities in the genus have also been detected 1973; Fig. 1b). Considering that this species is the only with nuclear data sets (Actine, 28S rDNA; Gonza´lez- kelp-dweller of the genus, its wide distribution could be Wevar 2010). For instance, Magellanic and Kerguele- attributed to long-distance dispersal by rafting (Donald nian subantarctic groups of the genus show high et al. 2005; Thiel & Gutow 2005; Waters 2007). levels of genetic divergence, supporting the existence of Recent analyses based on mitochondrial DNA agree physiological or dispersal constraints for Nacella larvae with previous morphological and molecular studies, and adults to long-distance dispersion. This result

2011 Blackwell Publishing Ltd CONCERTED EVOLUTION IN MAGELLANIC LIMPETS 1939 contrasts with the high levels of genetic affinity To evaluate these hypotheses, we measured the detected in different groups of marine taxa such as degree of genetic differentiation among seven taxa Macrocystis (Coyer et al. 2001), Mytilus (Ge´rard et al. (N. chiloensis, N. deaurata, N. flammea, N. fuegiensis, 2008), Durvillaea (Fraser et al. 2009, 2010, 2011), Sterechi- N. magellanica, N. mytilina and N. venosa). Based on the nus (Dıáz et al. 2011) and trochid gastropods (Donald mtDNA (cytochrome oxidase subunit I, COI), diversity et al. 2005) between distant provinces in the Southern pattern in sympatric Nacella nominal species, we pro- Ocean. Also, high levels of genetic divergence were pose to verify whether the nominal taxa of this genus detected between Nacella species from Antarctica and correspond to a single panmictic genetic unit in the the Magellanic Province, in contrast to the Antarctic– Magellanic Province with a broad distribution and a Magellanic connection described considering species wide range of habitat preferences or whether they cor- lists in several groups of marine benthic invertebrates respond to discrete genetic units. Predictions for these (Brandt et al. 1999; Crame 1999; Arntz 2005; Linse et al. hypotheses are as follows: (i) If Nacella nominal species 2006; Griffiths et al. 2009). In fact, the closest relative of correspond to different ecotypes of a single phenotypi- the Magellanic species of the genus is not the Antarctic cally variable species, we expect to find a lack of genetic limpet N. concinna, but N. clypeater from the Peruvian differentiation among the analysed units found in Province (Gonza´lez-Wevar et al. 2010a). Divergence sympatry. According to this, the existence of different time estimations using a relaxed Bayesian method sug- morphotypes, described as different species, would be a gest that the separation of Nacellidae (Nacella–Cellana) consequence of morphological changes associated with took place after the middle miocene climatic transition the different habitats where settlement and growth (MMCT) 14 Ma (Gonza´lez-Wevar et al. 2010a). This occur. In this case, nominal species described in this period was characterized by drastic climatic and ocean- region would correspond to morphotypes or ecotypes, ographic changes in the Southern Ocean (Flower & and the underlying mechanism would be phenotypic Kennett 1994; Zachos et al. 2001; Mackensen 2004; plasticity. Such a situation has been described in the Lewis et al. 2008; Verducci et al. 2009). Gonza´lez-Wevar Antarctic limpet N. concinna, where two very different et al. (2010a) proposed that the diversification of Nacella morphotypes (a shallow water form and a deeper water took place at least in two major rounds, long after the one) previously described as different species belong to separation of the Provinces that they currently inhabit the same genetic pool (Beaumont & Wei 1991; Hoffman along the Southern Ocean. The first round, at the end of et al. 2010; Gonza´lez-Wevar et al. 2011; but see de the Miocene (between 9.0 and 5.0 Ma) originated the Aranzamendi et al. 2008). (ii) Alternatively, if sympatric main lineages of the genus distributed in the Antarctic morphotypes associated with nominal species corre- and Kerguelenian provinces, as well as the separation spond to different reproductive units, we expect to of Nacella lineages in South America. The second corre- detect significant genetic differentiation among them, sponded to a recent Pleistocene diversification of the even if they originated recently on a mutational time genus (2.0 to 0.4 Ma) in the Magellanic Province, where scale. In this scenario, related species should share the different morphological species showed high levels of most common haplotypes but exhibit differences in genetic identity and an absence of reciprocal mono- their frequencies because of genetic drift. In this case, phyly (de Aranzamendi et al. 2009; Gonza´lez-Wevar the different genetic units found in sympatry could cor- et al. 2010a). However, previous studies in the genus respond to true biological species, and the underlying analysed a limited number of Magellanic nominal spe- mechanism for morphological variation would be adap- cies of Nacella and a low number of specimens. Based tive processes associated with niche differentiation. We on these preliminary results, the extremely short also performed geometric morphometric comparisons in branches and the lack of reciprocal monophyly among the analysed specimens to determine the degree of dif- Magellanic species of Nacella could be explained by two ferentiation among them to compare these results with different hypotheses. First, the previously described the genetic information. Nacella species in this Province correspond to a single morphologically variable species with multiple habitat Materials and methods affinities. Considering that these Magellanic morphospecies occur in sympatry and exhibit ecological differ- Sampling and specimen identification ences they could even be interpreted as ecotypes. Alternatively, the high number of Nacella species in this Magellanic species of Nacella were identified based on Province could reflect a very recent diversification pro- shell morphology and diagnostic external characters fol- cess in this region followed by rapid morphological and lowing Powell (1973) and Valdovinos & Ru¨ th (2005). ecological differentiation as proposed by Gonza´lez- We included in the analyses Nacella nominal species Wevar et al. (2010a). that clearly constitute different units such as N. deaurata,

2011 Blackwell Publishing Ltd 1940 C. A. GONZA´ LEZ-WEVAR ET AL.

N. flammea, N. magellanica and N. mytilina. We also random iterations) of haplotype identities to confirm incorporated N. chiloensis, N. fuegiensis and N. venosa, the presence of differences among the analysed units. whose taxonomic status is still unclear (Valdovinos & The comparisons of these indices will let us determine Ru¨ th 2005). To avoid genetic variation because of geo- whether the described morphospecies of Nacella from graphical distribution, we obtained samples of N. deau- Punta Santa Ana and Pelluco are distinct genetic units. rata, N. flammea, N. fuegiensis, N. magellanica and We also performed AMOVA analyses in Arlequin to N. mytilina from intertidal and subtidal zones of the determine the partition that maximizes the differences same locality, Punta Santa Ana (5337¢58¢¢LS; among groups. 7054¢50¢¢LW), Magellan Strait. We also collected sam- We reconstructed genealogical relationships among ples of N. chiloensis and N. venosa from Pelluco, Puerto Nacella units using median-joining haplotype networks Montt (4128¢51¢¢LS; 7254¢11¢¢LW; Fig. 1) because of its with Network 4.5.1.0 (Ro¨hl 2002). This method allows northern distribution in the Magellanic Province. In the reconstruction of phylogenies based on intra- and spite of the distribution ranges specified by Powell interspecific molecular data such as mitochondrial (1973), none of the other Nacella morphospecies were DNA variation. Haplotype network analyses were car- found in this area. ried out individually for each morphospecies, and we also constructed an overall network with all analysed individuals. DNA Extraction, PCR amplification and alignment Animals were fixed in ethanol (95%), and total DNA Geometric morphometrics analyses was extracted from mantle tissue using the salting-out protocol (Aljanabi & Martinez 1997). Shells of all the Shell-shape variation among Magellanic Nacella mor- specimens were kept for further geometric morphomet- phospecies from Punta Santa Ana and Pelluco was mea- ric analyses. A partial fragment of the mtDNA gene sured using outline analyses based on elliptic Fourier cytochrome C oxidase subunit I (COI) was amplified analyses. Outlines were drawn from digital photo- using universal primers described by Folmer et al. graphs using a two-dimensional projection of the lateral (1994). PCR amplifications were performed following shape of the shells of the same specimens used in the Gonza´lez-Wevar et al. (2010a); amplicons were purified genetic comparisons. Only adult specimens (>4 cm) using QIAquick Gel Extraction Kit (QIAGEN) and were included in the analyses. Elliptic Fourier transfor- sequenced in both directions. All haplotype sequences mations were made using SHAPE software (Iwata & were deposited in GenBank under the Accession Num- Ukai 2002). Elliptic Fourier descriptors (EFDs) can be bers HQ997162–HQ997364. used to depict any kind of form and have been effec- Sequences were edited with Proseq 2.91 (Filatov 2002) tively applied to the estimation of different shapes in and aligned with Clustal W (Thompson et al. 1992). plants and animals (Iwata & Ukai 2002). The method is Using MEGA 5.0 (Kumar et al. 2008), COI sequences were based on separate Fourier decompositions of the incre- translated into amino acids to check for sequencing mental changes of the x and y coordinates as a function errors and ⁄ or the presence of pseudogenes. of the cumulative length along the outline (Renaud & Michaux 2007). With the module ChainCoder, we extracted the contours of the objects and the relevant Genetic comparisons among Magellanic species of information was stored as chain codes. We obtained the Nacella normalized EDSs from the chain-coded contour with We determined the levels of polymorphism in the Mag- the module Chc2Nef, and the coefficients of the EFDs ellanic morphotypes of Nacella using standard molecu- were calculated by discrete Fourier transformation fol- lar indices such as the haplotype number (k), haplotypic lowing Kuhl & Giardina (1982). These coefficients were diversity (H), the number of segregating sites (S), aver- normalized, based on the ellipse of the first harmonic, age pairwise sequence differences (P) and nucleotide to be invariant with respect to size, rotation and starting diversity (p) with DnaSP 5.00.07 (Librado & Rozas point. With the module PrinComp, we performed the 2009). For comparison purposes, we also reconstructed principal components analyses on the variance–covari- the distribution of pairwise differences among Nacella ance matrix of the EFD coefficients. Principal compo- taxa. nents compile the information of the variation We estimated the levels of genetic differentiation contained in the coefficients (Rohlf & Archie 1984) and between the analysed units through mean pairwise dif- were estimated using PAST v.1.77 (Hammer et al. ferences among groups (NST) and through their haplo- 2001). Multivariate analysis of variance (MANOVA) was type frequencies (GST) in Arlequin v.3.11 (Excoffier performed with PAST to evaluate the importance of et al. 2005). We performed permutation tests (20 000 between-group differentiation relative to within-group

2011 Blackwell Publishing Ltd CONCERTED EVOLUTION IN MAGELLANIC LIMPETS 1941 variation. We performed Hotelling paired comparisons ular studies in Nacella (de Aranzamendi et al. 2009; tests to determine the significance of the morphological Gonza´lez-Wevar et al. 2010a; Gonza´lez-Wevar et al. differences. Finally, to estimate the percentage of cor- 2010b). rectly re-assigned individuals in each of the analysed Results obtained from the comparisons between Mag- nominal species, we used the information contained in ellanic nominal species of Nacella exhibited high levels the principal components of shell morphology in a mul- of genetic similarity. Only 50 characters were variable tivariate discriminant analyses with Statistica V.7.0.61.0 (7.4%), and 26 were parsimony informative (3.8%). (StatSoft Inc 2004). Genetic diversity indices such as the number of polymorphic sites, haplotype and nucleotide diversities were variable among morphospecies (Table 2). Haplo- Results type diversity varied among the analysed units with N. mytilina exhibiting the lowest value (0.392), while Molecular analyses N. flammea showed the highest one (0.892; Table 2). The COI sequence data set of Magellanic Nacella nomi- The number of haplotypes and polymorphic sites also nal species included 658 bp amplified from 208 individ- varied among species with N. mytilina showing the uals. We included 163 individuals belonging to lowest values even when we analysed the double num- N. deaurata (n = 25), N. fuegiensis (n = 28), N. magella- ber of specimens in this nominal species compared to nica (n = 25), N. flammea (n = 31) and N. mytilina the other ones. Finally, the average number of nucleo- (n = 54) from Punta Santa Ana, Magellan Strait, and 45 tide differences (P) and nucleotidic diversity (p) were specimens of N. chiloensis (n = 23) and N. venosa low and variable between morphospecies (Table 2).

(n = 22) from Pelluco, Puerto Montt (Table 2). As GST and NST paired comparisons between the differ- expected with coding regions, no indels or stop codons ent morphological units from Punta Santa Ana showed were detected in the complete data set, and we detected significant differences (P < 0.001, Table 3), except for one substitution, from leucine (L) to phenylalanine (F), the comparison between N. deaurata and N. fuegiensis. both nonpolar and neutral. This change corresponded In samples from Pelluco, no significant genetic differ- to a transition from C to T in the third position of ences were found between N. venosa and N. chiloensis. codon 85. Sequences were A–T rich (60.5%) compared However, both nominal species showed significant to the C–G content (39.5%), as observed in other molec- genetic differences with all other units from Punta

Table 2 Number of individuals per nominal species and their respective diversity indices based on mtDNA (COI) sequences

Morphospecies nkH SPp n MG

N. chiloensis 23 12 0.814 11 1.336 0.0019 22 N. deaurata 25 10 0.780 12 2.980 0.0047 25 N. ﬂammea 31 15 0.892 17 2.963 0.0044 30 N. fuegiensis 28 7 0.611 10 2.794 0.0041 25 N. magellanica 25 12 0.879 17 2.374 0.0035 22 N. mytilina 54 8 0.392 8 0.559 0.0008 30 N. venosa 22 11 0.827 11 1.459 0.0021 20 n, analysed specimens; k, haplotype number; H, haplotypic diversity; S, polymorphic sites; P, average nucleotide differences; p, nucleotidic diversity; n MG, number of individuals included in the geometric morphometric analyses.

Table 3 GST (below the diagonal) and NST (above the diagonal) pairwise comparisons among the analysed morphospecies of Nacella. 20 000 iterations. Statistically signiﬁcant differences are marked in bold

Morphospecies 1 2 34567

1. N. chiloensis — 0.205 0.320 0.281 0.013 0.778 0.032 2. N. deaurata 0.171 — 0.173 0.022 0.104 0.681 0.203 3. N. ﬂammea 0.118 0.145 — 0.176 0.228 0.577 0.315 4. N. fuegiensis 0.290 0.003 0.231 — 0.174 0.694 0.278 5. N. magellanica 0.011 0.124 0.089 0.239 — 0.715 0.016 6. N. mytilina 0.438 0.449 0.378 0.517 0.417 — 0.773 7. N. venosa 0.035 0.166 0.113 0.285 0.086 0.434 —

2011 Blackwell Publishing Ltd 1942 C. A. GONZA´ LEZ-WEVAR ET AL.

Santa Ana, with the exception of their putative species, AMOVA analyses. A total of fifteen haplotypes were N. magellanica (Powell 1973). AMOVA analyses suggest detected in Nacella flammea with a dominant haplotype that the partition best explaining the variance among (H32; 39%) and several haplotypes in low frequency groups included the following relationships: group (i) (Fig. 2c). As observed in Table 3, N. mytilina had the N. chiloensis—N. magellanica—N. venosa; group (ii) lowest levels of genetic diversity with 78% of the indi- N. deaurata—N. fuegiensis; group (iii) N. flammea; and viduals sharing the same haplotype (H24; Fig. 2d). group (iv) N. mytilina, corroborating GST and NST Dominant haplotypes in N. flammea (H32; Fig. 2c) and paired comparisons. In this partition, variation among N. mytilina (H24; Fig. 2d) were only present in these groups explained more than 30% of the genetic vari- nominal species. ance, while variation among morphospecies within Nacella nominal species can be assigned to three differ- groups accounted for <1%. ent patterns of pairwise difference distribution, further Individual median-joining network analyses resulted supporting the high levels of genetic affinities among in star-like (N. chiloensis, N. magellanica, N. mytilina and these units (Fig. 2). First, an L-shaped distribution char- N. venosa) and more expanded (N. deaurata, N. fuegien- acterized N. mytilina (Fig. 2d), as expected in a star-like sis and N. flammea) genealogies (Fig. 2). The genealo- genealogy. Second, a unimodal pattern of distribution gies of N. deaurata (Fig. 2a) and N. fuegiensis (Fig. 2b) characterized N. flammea (Fig. 2c), N. magellanica were similar, sharing several haplotypes including the (Fig. 2e), N. chiloensis (Fig. 2f) and N. venosa (Fig. 2g). dominant one (H1). A second group of haplotypes, Finally, a bimodal distribution distinguished the nominal separated by at least four mutational steps, was species N. deaurata (Fig. 2a) and N. fuegiensis (Fig. 2b). detected in both nominal species (H5 and H8; Fig. 2a, The overall median-joining network resulted in a com- b). Similarly, the genealogies of N. chiloensis (Fig. 2e), plex genealogy representing the high levels of genetic N. magellanica (Fig. 2f) and N. venosa (Fig. 2g) were similarity among them (Fig. 3). We recovered 53 haplo- also very similar, with a dominant haplotype (H2) types; at least three of them (H1, H2 and H24) at high shared among these three units. These results support frequency (14.8%, 17.2% and 20.3%, respectively). the absence of differentiation detected among these H1 was present in four morphospecies (N. deaurata, units in the pairwise GST and NST comparisons and in N. fuegiensis, N. magellanica and N. flammea), while H2

0.4 Expected Expected Observed Expected 0.2 Observed 0.2 Observed 0.3 H37 N. deaurata 0.2 N. fuegiensis N. flammea 0.1 0.1 0.1 H9 H34 0 10 20 H9 10 20 10 20 Pairwise differences H7 Pairwise differences Pairwise differences H10 H5 H2 H1 mv1 mv2 H35 H41 H5 H8 H8 H31 H1 H11 H32 H39 H36 H2 H10 H1 H12 H40 H23 H38 H3 H4 H33 H6 H13

Expected 0.4 Expected 0.6 Expected Expected Observed Observed Observed Observed 0.3 0.2 0.3 0.4 N. chiloensis 0.2 N. venosa N. mytilina N. magellanica 0.2 0.1 0.2 0.1 0.1

10 20 10 20 10 20 10 20 Pairwise differences Pairwise differences H18 Pairwise differences Pairwise differences H49 H51 H22 H46 H25 H28 H47 H52 H21 H53 H26 H46 H45 H2 H17 H2 H50 H1 H2 H44 H20 H48 H44 H49 H24 H29 H15 H20 H14 H20 H42 H42 H23 H9 H27 H30 H43 H16 H9 H43 1 Substitution

40 20 10 1

Fig. 2 Individual haplotype network and pairwise mismatch distribution in seven nominal species of Nacella from Punta Santa Ana and Pelluco. (a) N. deaurata—red; (b) N. fuegiensis—green; (c) N. ﬂammea—purple; (d) N. mytilina—blue; (e) N. magellanica—yellow; (f) N. chiloensis—light blue; (g) N. venosa—orange. Each haplotype is represented by a coloured circle whose size is proportional to its frequency, mv are median vectors or hypothetical haplotypes.

2011 Blackwell Publishing Ltd CONCERTED EVOLUTION IN MAGELLANIC LIMPETS 1943

H25 Fig. 3 Overall haplotype network H26 including seven nominal species of H38 Nacella. Each haplotype is represented H29 H27 H17 by a coloured circle whose size is H24 proportional to its frequency. mv corre- H30 sponds to median vectors or hypotheti- H34 H3 cal haplotypes. Red, N. deaurata; green, H6 H15 H41 H4 N. fuegiensis; purple, N. ﬂammea; blue, H28 N. mytilina; yellow, N. magellanica; light H18 H23 H10 blue,N. chiloensis;orange,N. venosa. H1 H13 H39 H12 H32 H36 H11 H8 H53 H47 mv01 H37

H51 H2 H31 H49 H5 H50 H43 H42 H35 H48 H40 H21 H52 H20 H46 H33 H14 H7 H19 H22 H44

H9 H16 H45

Nacella chiloensis Nacella magellanica 1 Substitution 40 30 Nacella deaurata Nacella mytilina 10 Nacella flammea Nacella venosa 1 Nacella fuegiensis was present in five (N. chiloensis, N. deaurata, PC1 and PC2 explained 91.37% of the variance; PC1 N. flammea, N. magellanica, N. venosa; Fig. 3). It is (80.45%) consisted of differences in shell height while important to indicate that H1 was more frequent in PC2 (10.92%) corresponded to changes in the lateral N. deaurata and N. fuegienesis, while H2 was more abun- ends of the shells (Fig. 4). MANOVA analyses detected dant in N. magellanica, N. chiloensis and N. venosa. significant differences among the analysed units (Wilks’ N. chiloensis and N. venosa shared the dominant haplo- k 0.06693; P < 0.0001). However, several of them were type of their putative parental species N. magellanica clearly overlapped, such as N. deaurata–N. fuegiensis, (H2) and not the dominant haplotype of N. deaurata. N. chiloensis–N. venosa, N. chiloensis–N. magellanica, and These results further support the absence of genetic N. venosa–N. magellanica (Fig. 4). Hotelling paired com- differentiation between N. deaurata and N. fuegiensis parisons detected significant differences in all compari- and among N. magellanica, N. chiloensis and N. venosa. sons, with the exception of those between N. fuegiensis– As stated earlier, a dominant haplotype (H24) was only N. deaurata, N. chiloensis–N. venosa, N. magellanica– present in N. mytilina (Fig. 3). We also detected some N. chiloensis, and N. magellanica–N. venosa (Table 4). haplotypes at medium frequencies (H5, H9, H23) shared Multivariate discriminant analyses based on shell mor- by five or two morphospecies. H9 was found in N. chilo- phologies among the nominal species of Nacella are in ensis, N. deaurata, N. flammea, N. magellanica and basic agreement with our geometric morphometric N. venosa; H5 was found in N. deaurata and results (Table 5). For instance, we detected low levels N. fuegiensis, and H23 was present in N. flammea and of correct re-assignment (<45%) in the nominal species N. mytilina. The rest of the haplotypes were found at N. magellanica, N. chiloensis and N. venosa. Similarly, low frequency and in most cases were specific to the low levels of correct re-assignment were detected in analysed nominal species (Fig. 3). N. deaurata and N. fuegiensis (<55%). In contrast, high levels of correct re-assignment were observed in N. mytilina (100%) and in N. flammea (93%). Considering our Geometric morphometrics morphometric and genetic results, we performed a new Principal components analysis of the shell morphology discriminant multivariate analysis including only four showed marked differences among the analysed units. groups: (i) N. magellanica ⁄ N. chiloensis ⁄ N. venosa; (ii)

2011 Blackwell Publishing Ltd 1944 C. A. GONZA´ LEZ-WEVAR ET AL.

Fig. 4 Multivariate principal components analysis of the shell-shape variation in the analysed sympatric nominal species of Nacella from Punta Santa Ana and Pelluco. Red, N. deaurata; Light green, N. fuegiensis; Purple, N. ﬂammea; Blue, N. mytilina; Yellow, N. magellanica; Light blue, N. chiloensis; Green, N. venosa.

Table 4 P-values of the paired Hotelling test (Bonferroni corrected) of the PC analyses obtained from the morphology of the shells of different Magellanic Nacella nominal species. Signiﬁcant comparisons are indicated in bold

Morphospecies 1234567

1. N. chiloensis — 2. N. deaurata <0.001 — 3. N. ﬂammea <0.001 <0.001 — 4. N. fuegiensis <0.001 0.221 <0.001 — 5. N. magellanica 0.967 <0.001 <0.001 <0.001 — 6. N. mytilina <0.001 <0.001 <0.001 <0.001 <0.001 — 7. N. venosa 0.348 <0.001 0.278 <0.001 <0.001 <0.001 —

Table 5 Multivariate discriminant analyses based on principal components (PC1 and PC2) among nominal species of Nacella in the Magellanic Province considering the percentage of correctly re-assigned individuals and their corresponding frequencies

Percent correctly re-assigned 1 2 3 4 5 6 7

1. N. magellanica 41 975 0 0 0 1 2. N. chiloensis 36 388 0 0 0 3 3. N. venosa 40 758 0 0 0 0 4. N. mytilina 100 0 0 0 30 0 0 0 5. N. ﬂammea 93 000 1281 0 6. N. deaurata 52 0 1 1 0 0 13 10 7. N. fuegiensis 48 2 1 0 0 0 10 12

N. deaurata ⁄ N. fuegiensis; (iii) N. mytilina; (iv) and parisons among the analysed nominal species of Nacella N. ﬂammea. Discriminant analyses in these new data set in the Magellanic Province. detected high percentage of correct re-assignment of individuals (>85%; Table 5). According to these results, Discussion we distinguished four morphological groups in the analysed nominal units of Nacella: (i) N. deaurata– In this study, we present new genetic and morphologi- N. fuegiensis, (ii) N. magellanica–N. chiloensis–N. venosa, cal data on the relationship among seven out of the (iii) N. mytilina, and (iv) N. ﬂammea. These morphologi- eight previously described species of Nacella in the Mag- cal results are in total agreement with our genetic com- ellanic Province. We did not consider N. delicatissima

2011 Blackwell Publishing Ltd CONCERTED EVOLUTION IN MAGELLANIC LIMPETS 1945 as a separate species because de Aranzamendi et al. geometric morphometric Hotelling tests and with the (2009), using ISSR, concluded that N. delicatissima corre- correct re-assignment of individuals in the discriminant sponds to an ecotype of N. magellanica or N. deaurata. analyses. As for COI comparisons, no differences were Our genetic results further expand previous phyloge- detected among N. chiloensis, N. venosa and N. magella- netic (Gonza´lez-Wevar et al. 2010a) and phylogeograhic nica, or between N. fuegiensis and N. deaurata. Finally, studies (de Aranzamendi et al. 2009) of the group, four genetic and morphological groups, each associated establishing the monophyly of all the analysed Magel- with a different habitat, were detected in Nacella from lanic units. Nominal species of Nacella in this Province the Magellanic Province. appear very closely related and several haplotypes were shared among them. This high level of genetic similar- High levels of genetic similarity in Magellanic nominal ity has also been detected using nuclear markers such species of Nacella as Actine and 28S rDNA, which showed an absence of variation among all the Magellanic units (Gonza´lez- Absence of reciprocal monophyly and elevated levels of Wevar 2010). In contrast, when compared with Nacella genetic identity have been reported in newly formed species from different biogeographical Provinces of the species of different taxa such as cichlid fishes (Meyer Southern Ocean, these markers exhibited notable levels et al. 1990; Meyer 1993; Verheyen et al. 2003; Schliewen of divergence (Gonza´lez-Wevar 2010). et al. 2004), finches (Sato et al. 1999; Edwards et al. Our COI results indicate that the five sympatric nom- 2005), spiny lizards (Wiens & Penkrot 2002), Moorean inal species from Punta Santa Ana correspond to four tree snails (Lee et al. 2009) and Hawaiian swordtail differentiated genetic units or divisions. Nacella deaurata crickets (Shaw 2002). Similar results as the one detected and N. fuegiensis did not show a significant level of in Nacella have also been detected in invertebrates and genetic difference, while all the other morphotypes seg- especially in mollusks. For instance, Sauer & Hausdorf regated individually. These results discard the hypothe- (2010) recognized a recent nonadaptive radiation trig- sis that all morphotypes found in sympatry correspond gered by sexual selection that resulted in the origin of to simple ecotypes of a single species. It is worth noting 10 endemic land snail species of Xerocrassa. Even when that each of these four genetic units is found in a differ- these species were clearly discriminated based on mor- ent habitat. For instance, N. magellanica inhabits high phological features, only five of them were monophy- and middle intertidal areas, while N. flammea inhabits letic in a mitochondrial gene tree. However, when all the subtidal zone (Powell 1973; Valdovinos & Ru¨ th these species were analysed using a multilocus 2005; C.A. Gonza´lez-Wevar, personal observation). The approach based on AFLPs markers, at least nine mono- two nominal species that did not show genetic differen- phyletic groups were recognized. In general, recently tiation, N. deaurata and N. fuegiensis, both inhabit the formed species exhibit high levels of morphological, low intertidal zone. Finally, N. mytilina, the species behavioural and ecological differences, providing evi- showing the highest pairwise GST and NST values corre- dence for adaptive radiation. This kind of process sponds to a subtidal species living exclusively over the occurs when divergent selection operates on characters giant kelp Macrocystis pyrifera. In the case of the Pelluco that favor different genetic variants, or alleles, in con- site from the northernmost part of the Magellanic Prov- trasting environments (Schluter 2000, 2001). Two main ince, pairwise genetic comparison between N. chiloensis speciation models have been proposed to explain the and N. venosa indicated absence of genetic differentia- co-occurrence of newly formed species (Sobel et al. tion between them. This absence of genetic structure is 2010) and they can be evoked to understand the recent again associated with similar habitat preferences, with diversification of Nacella in the Magellanic Province. both nominal species inhabiting middle and high inter- The first model corresponds to sympatric speciation, the tidal zones. Compared with individuals from Punta split of one population into two or more species in the Santa Ana, N. chiloensis and N. venosa showed signifi- absence of geographical isolation. A simplistic scenario cant genetic differences with all the other nominal spe- for this model involves disruptive selection in the habi- cies with the exception of N. magellanica. It is worthy to tat or mate choice, where natural selection drives a pop- note that N. magellanica, N. chiloensis and N. venosa cor- ulation in two different evolutionary trajectories respond to conspicuous and dominant invertebrates of (Dieckmann & Doebeli 1999; Doebeli & Dieckman 2003; the middle and high intertidal zones in both localities. Bolnick & Fitzpatrick 2007; Coyne 2007). This speciation The geometric morphometric and the multivariate model remains as one of the most contentious concepts discriminant analyses of this study are fully congruent in evolutionary biology because even when sympatric with the genetic results. All statistically significant speciation is theoretically possible, empirical studies are paired comparisons using the genetic differentiation scarce and few examples exist (Coyne & Orr 2004; Bar- indices GST and NST tests were also significant with luenga et al. 2006; Rolań-Alvarez 2007; Herder et al.

2011 Blackwell Publishing Ltd 1946 C. A. GONZA´ LEZ-WEVAR ET AL.

2008). The second model that may account for the co- structuring the present-day marine invertebrate assem- ocurrence of newly formed species involves geographi- blages in response to glacial advances, temperature cal isolation among populations followed by a second- shifts and sea level changes. For instance, marine ben- ary contact (Schluter 2001; Coyne & Orr 2004; Rundle & thic fauna currently found in the Magellan Strait is Nosil 2005). In the absence of gene flow, reproductive believed to have recolonized this area from adjacent isolation arises gradually as a result of mutation, genetic Atlantic and Pacific refugia (Montiel et al. 2005). In this drift and natural selection driven by local environmen- context, fragmentation and isolation of marine benthic tal conditions (Hoskins et al. 2005; Rundle & Nosil invertebrates into refugia or microbasins (Antezana 2005). When the geographical barrier disappears, expan- 1999) during the coldest periods may have favored the sion of the distribution area of incipient species may diversification of species in the Magellan fjords and produce a secondary contact zone or even a broad dis- straits in allopatric conditions (Crame 1997; Valdovinos tribution overlap (Bolnick & Fitzpatrick 2007). If repro- et al. 2003). According to this, allopatric speciation, or ductive isolation is complete, competition among newly at least incipient separation, followed by geographical formed species may strengthen niche differentiation, re-expansion and ecological separation represents a permitting their coexistence in the same area (Schluter more plausible scenario to explain the current overlap- 2000). Alternatively, in the case of incomplete reproduc- ping distribution of Nacella species and their close tive isolation, reinforcement could enhance ecological genetic relationships than does the sympatric speciation segregation and produce speciation, the taxa becoming model. In this context, differences in the shape of the independent evolutionary entities (Hoskins et al. 2005; mismatch distribution among the four genetic units Gavrillets & Vose 2007; Gavrillets & Losos 2009). could also reflect differences in their demographic his- The southern tip of South America has been subjected tory. Thus, ecological speciation, the evolution of barri- to major climatic changes during the Quaternary by ers to gene flow resulting from ecologically based recurrent glacial cycles that markedly modelled the geo- divergent selection (Schluter 2000, 2001; Rundle & Nosil morphology of this region (McCulloch et al. 2000, 2005; Hendry et al. 2007; Langerhans et al. 2007) may 2005a,b; Coronato et al. 2009). Although glacial events be the main driving process behind the diversification in the Magellanic Province did not reach the extent and of Nacella in the Magellanic Province. magnitude that occurred in Antarctica, recurrent and Finally, the recent diversification of Nacella in the Mag- periodic continental ice cap advances and retreats have ellanic Province gives an interesting opportunity to deeply impacted this region, playing an important role understand further the evolution of benthic marine in shaping the contemporary distribution and diversity fauna during the Pleistocene glacial cycles in this region. of different taxa (Pre´moli et al. 2000; Xu et al. 2009; Fra- Future studies in Nacella in the Magellanic Province will ser et al. 2010; Lessa et al. 2010; Zemlak et al. 2010). For attempt to identify the underlying processes and mecha- example, an extensive ice cap covered the western slope nisms of speciation to evaluate the possibility of an of the Andes from 35 to 55S during the last glacial adaptive radiation in the genus. At the same time, eco- maximum (LGM) 21 kya (Rabassa & Clapperton 1990; logical experiments will try to determine the degree of Benn & Clapperton 2000; Thatje & Brown 2009), while morphological plasticity in Nacella moving individuals sea level was 125–135 m lower than it is today (Fair- into different habitats to determine the existence of banks 1989; Rabassa et al. 2005). During this period, the shell-shape variations. Also, further phylogeographical Magellan Strait was completely covered by sea ice, as studies will include fast-evolving markers, which should well as most of the shallow subtital zone along the Pat- provide a better understanding of contemporary popula- agonian Chilean coastline (Thatje & Brown 2009). The tion dynamics, gene flow patterns and processes along the gradual warming process that followed the LGM complex geography in the southern tip of South America explains much of the modern biogeographical pattern that includes channels, inlets, fjords and land masses. in this Province (Arntz 2005; Montiel et al. 2005; Ruzz- ante et al. 2006, 2008; Zemlak et al. 2010). Postglacial Systematic implications patterns of genetic diversity in several taxa indicate the existence of several independent Quaternary glacial Delimitation of species is fundamental for analysis in refugia east of the Andes (Kim et al. 1998; Ruzzante biogeography, ecology, macroevolution and conserva- et al. 2006; Himes et al. 2008) and west of the Andes tion biology (Sites & Marshall 2003, 2004). The advent within and outside the northern and western limits of of easy DNA-based technologies has facilitated the use the glaciers (Pre´moli et al., 2000; Pastorino & Gallo of genetic markers for species delimitations and assess- 2002; Zemlak et al. 2008, 2010). According to Crame ments of biodiversity (Hebert et al. 2003; Stoeckle 2003; (1997), range retractions into refugia followed by subse- Janzen 2004). In general, gene trees are used as a quent re-expansion have also played a crucial role in proxy to delimit boundaries among taxa based on two

2011 Blackwell Publishing Ltd CONCERTED EVOLUTION IN MAGELLANIC LIMPETS 1947 principal genetic thresholds, reciprocal monophyly and N. deaurata, N. delicatissima, N. magellanica and N. myti- the 10· rule (10 times greater genetic divergence lina were only found in late Pleistocene (<40 kya) and between than within species; Sites & Marshall 2004; Holocene deposits (Gordillo 1999; Aguirre 2003; Gordil- Hickerson et al. 2006; Knowles & Carstens 2007). lo et al. 2005; Aguirre et al. 2006, 2009; Ca´rdenas & Gor- According to Baum & Shaw (1995) and Hebert et al. dillo 2009). Using an integrative approach, we observed (2003), these two criteria permit clear species delimita- a complete concordance between morphological and tion based on the genealogical descent of the genome, molecular results among Nacella nominal species from in contrast to the difficult task of discriminating repro- the Magellanic Province. Along with differences in hab- ductive compatibilities. Nevertheless, these methodolo- itat and ecological preferences, genetic and morphologi- gies raise concerns about the use of thresholds applied cal characters identified four distinct groups of Nacella, to genetic data (Hudson & Coyne 2002; Hudson & Tur- contrasting with previous taxonomic revisions (Powell elli 2003; Moritz & Cicero 2004). Identification of species 1973; Valdovinos & Ru¨ th 2005). The absence of signifi- based on exclusivity criteria is often incongruent with cant morphological and genetic differences between identifications based on other sources of data, question- N. chiloensis and N. venosa did not support their spe- ing the accuracy of species boundaries (Sites & Marshall cific status recognized by Valdovinos & Ru¨ th (2005). 2004; Balakrishnan 2005; Knowles & Carstens 2007). In Moreover, no differences were detected between these particular, species identification in recently derived spe- two nominal species from Pelluco and N. magellanica cies encompasses special difficulties because of incon- from Punta Santa Ana, in spite of the geographical dis- gruence between gene trees estimated from a particular tance separating sampling localities. Valdovinos & Ru¨ th locus and the real species tree (Avise & Ball 1990; (2005), using shell-shape NMDS analyses, also detected Knowles & Carstens 2007; Knowles & Chan 2008). a morphological overlap among these three units but Under the reciprocal monophyly criterion, newly finally described N. magellanica, N. chiloensis and formed species tend to go unrecognized, because N. venosa as three different species based on debatable boundaries among them are not faithfully reflected in diagnostic characters. Specifically, some of the chosen their DNA sequences until ancestral polymorphism has characters such as shape, thickness and shell coloration been fully sorted (Hebert et al., 2003; Hickerson et al. are recognized as highly homoplasic in patellogastro- 2006; Carstens & Knowles 2007). Integrative taxonomic pods (Lindberg 2008) and particularly in Nacella (Beau- studies have helped to clarify the relationship between mont & Wei 1991; Nolan 1991; Morriconi & Calvo 1993; closely related species by using a combination of molec- de Aranzamendi et al. 2008, 2009; Gonza´lez-Wevar ular and morphometric data (Roe & Sperling 2007a,b; et al. 2011). According to our results, N. magellanica, Padial et al. 2010). In a revision of the lottid limpet Pa- N. chiloensis and N. venosa should be considered as a telloida complex, in which Patelloida pygmaea, P. heroldi single unit: N. magellanica (according to the Interna- and P. conulus were previously synonymized and trea- tional Code of Zoological Nomenclature ICZN; ted as a single species with different ecological morpho- Table 1). This proposition is more in accordance with types, Nakano & Ozawa (2005) concluded that each of Powell (1973), who recognized N. chiloensis and N. ven- these ecological forms corresponded to a true taxonomic osa as subspecies of N. magellanica. Similarly, N. deaura- unit. In another study of the lottid limpet, mitochon- ta and N. fuegiensis belong to the same genetic and drial and nuclear sequences revealed that Notoacmea morphological unit, with subtle differences in shell col- helmsi should be split into five species, each related to oration and ornamentation and therefore could be con- different habitats (Nakano & Spencer 2007). sidered as a single taxon: N. deaurata (according to the In Nacella, reciprocal monophyly was not observed ICZN; Table 1). Finally, significant genetic and morpho- among nominal species from the Magellanic Province metric differences as well as marked segregation of (de Aranzamendi et al. 2009; Gonza´lez-Wevar 2010; their habitats supported the specific status of N. mytili- Gonza´lez-Wevar et al. 2010a). According to these na and N. flammea (Powell 1973; Valdovinos & Ru¨ th results, these taxa could not be validated under the 2005). These results should encourage further integra- phylogenetic species concept. However, in recently tive systematic analyses in this cold-water genus includ- derived species it is expected that lineage sorting have ing multiple characters to encompass a profound not been fully completed, explaining why several spe- taxonomic revision of the group. cies share the most common haplotypes. This explanation may be invoked in Nacella, for which Pleistocenic Acknowledgements diversification has been previously proposed in the Magellanic Province (Gonza´lez-Wevar et al. 2010a). Fur- We are grateful to the following people and museums for help thermore, the fossil record of the genus in this region in field work, data analyses and for contributing specimens supports recent diversification. Specimens identified as to this study: Ce´sar Ca´rdenas, Alejandro Riedemann, Cristiań

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T.N.’s research interests are Taxon- StatSoft Inc (2004) STATISTICA (data analysis software omy and Systematics, Evolutionary Biology, biogeography, and system), version 7.0. www.statsoft.com. population genetics in several groups of patellogastropods and Stoeckle M (2003) Taxonomy, DNA, and the barcode of life. specially in Cellana. J.I.C.’s research interests are taxonomy and BioScience, 53, 796–797. biodiversity of polychaetes. He also works in reproductive Thatje S, Brown A (2009) The macrobenthic ecology of the patterns of marine invertebrates along the Humboldt Current Straits of Magellan and the Beagle Channel. Anales Instituto System and Cape Horn, benthic biodiversity in subantarctic Patagonia (Chile), 37, 17–27. marine protected areas. E.P.’s main research interests are the Thiel M, Gutow L (2005) The ecology of rafting in the marine origin and evolution of the marine benthic fauna in the South- environment. I. The floating substrata. Oceanographic Marine ern Ocean, with special emphasis in Antarctica. He also is Biology Annual Review, 42, 181–263. interested in Evolutionary Biology, biogeography, and popula- Thompson JD, Higgins DG, Gibson TJ (1992) Clustal W: tion genetics. improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap

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