Hidden Diversity and Cryptic Speciation Refute Cosmopolitan Distribution in Caprella Penantis (Crustacea: Amphipoda: Caprellidae)

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1Laboratorio de Biología Marina, Departamento de Zoología, Facultad de Biología, Universidad de Sevilla, Seville Spain; 2Museo Nacional de Ciencias Naturales (MNCN, CSIC), Madrid Spain; 3 Present address: Department of Biology, Brigham Young University, 401 Widtsoe Building,Provo UT, 84602, USA Hidden diversity and cryptic speciation refute cosmopolitan distribution in Caprella penantis (Crustacea: Amphipoda: Caprellidae)

1 2,3 2 1 MARÍA PILAR CABEZAS ,PATRICIA CABEZAS ,ANNIE MACHORDOM and JOSÉ M. GUERRA-GARCÍA

Abstract Caprella penantis is considered a cosmopolitan species and one of the most challenging caprellids in taxonomic terms because of its remarkable intraspecific morphological variation. This study examined DNA sequences from mitochondrial (COI) and nuclear (18S) markers together with morphological data from 25 localities of C. penantis, and closely related species Caprella dilatata and Caprella andreae, all traditionally considered part of the old ‘acutifrons’ complex. The large genetic divergence and reciprocally allopatric distributions point to the existence of a species complex of at least four species, of which one is reported as a cryptic species. This study provides the first evidence of cryptic speciation in the family Caprellidae, and questions the validity of some traditional morphological characters used to delimit species in the genus Caprella. Our results are consistent with the idea that main factors were probably isolation by distance and ecological traits, promoting diversification in C. penantis. The strong genetic structure reported for this species in the Iberian Peninsula and Moroccan coasts also suggests restriction to dispersal as well as the presence of refugial areas. These results highlight the utility of the COI and 18S genes in combination with morphological characters for shedding light on systematic questions in caprellids, and patterns of genetic connectivity.

Key words: COI – cryptic species – genetic structure – morphology – 18S – taxonomy

Introduction gammarids (e.g. Hogg et al. 2006; Witt et al. 2006; Seidel et al. 2009; Pilgrim and Darling 2010; Baird et al. 2011). Particularly Scientists have long been interested in understanding the ecologi- interesting is the species Caprella penantis Leach, 1814 (Crus- cal and evolutionary processes underlying the origin, distribution tacea, Amphipoda, Caprellidae), which despite its limited power and preservation of biological diversity, with increasing attention of dispersal, is considered to have a cosmopolitan distribution during recent years because of the enormous loss of worldwide covering tropical, subtropical and temperate oceans (McCain diversity (Costello et al. 2010). Mitigation measures are required, 1968; Vassilenko 1991; Krapp-Schickel 1993). Caprella penan- but difficulties arise due to the unknown extent of biodiversity tis constitutes one of the dominant caprellid species in intertidal and spatial distribution of species assemblages (Witt et al. 2006; communities and shallow waters in marine ecosystems (Guerra- Radulovici et al. 2009), as well as the difficulty describing spe- García 2001; Guerra-García et al. 2009b,c; Guerra-García and cies based solely on morphological characters (Knowlton 1993; Izquierdo 2010), and an important dietary component for many Remerie et al. 2006; Beheregaray and Caccone 2007). In the coastal marine fish species (Caine 1989; Woods 2009). It can marine environment, this fact is even more remarkable because À reach densities higher than 10 000 individuals m 2 in intertidal genetic and ecological studies frequently highlight the existence seaweeds of temperate ecosystems (Guerra-García et al. 2009c, of cryptic species (Knowlton 1993, 2000; Mathews 2006; Calvo d, 2010a; Guerra-García and Izquierdo 2010) and can go et al. 2009): species that are genetically distinct, but difficult to À beyond 50 000 individuals m 2 in other marine habitats (see distinguish using morphological characters (Mayr 1948, 1963; Takeuchi 1999). Moreover, it has recently been considered a Mayr and Ashlock 1991). Unclear species boundaries and cryptic sensitive species (Guerra-García and García-Gomez 2001) as speciation are common problems in a wide range of marine well as a good biomonitor of trace metal contamination in these organisms (Knowlton 1993; Avise 1994; Witt et al. 2006). ecosystems, even better than other marine invertebrates (Guerra- Establishing species boundaries, involving the identification of García et al. 2009a, 2010b). Despite its abundance, cosmopoli- cryptic species, is fundamentally important in biodiversity assess- tan distribution and importance as bioindicator, the taxonomy of ment (Knowlton 1993, 2000; Cook et al. 2008) and in subse- C. penantis remains unsettled (Mayer 1890, 1903; McCain and quent conservation strategy design (Bickford et al. 2007). Steinberg 1970). The taxonomy status of this species has been Reliance on morphology-based taxonomy alone might critically fraught with controversy for years, and it has been recorded underestimate biodiversity. Consequently, combination of multi- under several species or subspecies names from temperate ple types of independent sources of data, including molecular, regions worldwide (McCain and Steinberg 1970) because of its morphological and ecological data, is required to accurately considerable intraspecific morphological variability (McCain assess species boundaries (Remerie et al. 2006; Roe and Sperling 1968; Laubitz 1972; Guerra-García et al. 2006; Cabezas et al. 2007; Hou and Li 2010). 2010). In his monographs, Mayer (1890, 1903) described 19 Among crustaceans, the order Amphipoda is one of the most forms of the ‘Caprella acutifrons’ group. Several of these forms problematic taxonomic groups because of the difficulty to iden- have already been given specific rank (Utinomi 1943; Dougher- tify diagnostic characters (Martin and Davis 2001; Browne ty and Steinberg 1953; Vassilenko 1967; Laubitz 1972). For et al. 2007), and cryptic speciation has been widely reported for example, form andreae was assigned to Caprella andreae Mayer, 1890 (McCain 1968); forms typica and minor have been Corresponding author: María Pilar Cabezas Rodríguez ([email protected]) assigned to Caprella dilatata Krøyer, 1843 (McCain 1968) and Contributing authors: Patricia Cabezas ([email protected]), probably forms tabida and tibada also belong to this species, Annie Machordom ([email protected]), Jose M. Guerra-García although their taxonomic status under C. penantis is still under ([email protected])

J Zoolog Syst Evol Res (2013) 51(2), 85--99 86 CABEZAS,CABEZAS,MACHORDOM and GUERRA-GARCÍA discussion (Guerra-García et al. 2006). Forms gibbosa, carolin- code and coordinates for the sampling localities, as well as some collec- ensis, virginia, lusitanica, testudo and simulatrix are still classi- tion information are summarized in Table 1. fied under the species C. penantis (McCain 1968; Laubitz 1970, 1972; Krapp-Schickel 1993), characterized mainly by the pres- Morphological analyses ence of short and triangular rostrum on the front of the head, second gnathopods with proximal poison tooth and pereopods 5 All individuals were morphologically identified by stereomicroscope –7 propodi palm slightly concave with proximal grasping according to the characters described by Mayer (1882, 1890, 1903) and spines. Under this framework, it is clear that more comprehen- Krapp-Schickel (1993). Only adult males were considered, as most of the fi sive studies are urgently needed to distinguish between intra- species-speci c diagnostic characters are fully developed and more obvious in these specimens. We particularly examined male gnathopods, gill shape, and interspecific variability within C. penantis. robustness of antenna 1, concavity/convexity of propodi palm in pereopods Like all peracarid crustaceans, the dispersion of C. penantis is 5–7 (P5-7), presence/absence of ‘grasping spines’ and body length. assumed to be primarily driven by rafting (Thiel 2002; Thiel et al. 2003b) because of the lack of a planktonic larval stage (Thiel and Vasquez 2000; Cook et al. 2004) and limited swim- DNA extraction, PCR amplification and sequencing ming capabilities (Thiel et al. 2003b). Thus, considerable genetic Genomic DNA was extracted from legs, gnathopods, gills and pereopods differentiation among populations and the existence of cryptic except when individuals were too small (in those cases, the whole speci- species is expected. Here, we present the first study to clarify men was used) using the BioSprint 15 DNA Blood Kit (45) (Qiagen the taxonomic status of the species C. penantis as well as evalu- Iberia, Madrid, Spain). Protocol was carried out according to the manu- ate genetic connectivity patterns in relation to life history and facturer’s instructions, but with elution volume decreased to 100 llto ecological traits. For this purpose, we examined genetic variabil- maximize DNA concentration. Final DNA concentration was estimated ity in mitochondrial and nuclear genes together with morphologi- using a Nanodrop Spectrophotometer (Nanodrop 1000: Thermo Scientific, cal characters in 30 populations across its cosmopolitan Madrid, Spain). distribution. An approximately 1200-base pair (bp) fragment of the mitochondrial DNA (mtDNA) cytochrome c oxidase subunit 1 gene (COI) was amplified in two overlapping PCR fragments of 550 and 660 bp respectively. Material and methods Because of the poor amplification success of the universal COI primers, LCO1490 and HCO2198 (Folmer et al. 1994) and COI2F and COI2R Sample collection (Otto and Wilson 2001), some sets of genus-specific COI primers were designed (Table 2) using the software package PRIMER3 (Rozen and Ska- A total of 105 specimens belonging to 30 populations of C. penantis letsky 2000). The primer pairs COIF2/COIR1 and COIF3/COI2R were were collected. Populations of the morphologically closely related species both the most successful in the amplification of the first and second C. dilatata (6) and C. andreae (7) were also included as a reference for amplicons respectively. However, some samples failed the amplification interspecific genetic divergence among congeneric species (Fig. 1). Speci- with the above primer combinations, so that we also designed one set of mens were preserved in 95–100% ethanol for subsequent molecular anal- species-specific primers for each fragment (Table 2). For all sets of yses. Information about all individuals included in this study, population

Fig. 1. Map of sampling localities for Caprella penantis (●) and the closest species Caprella andreae ( ) and C. dilatata (■) included in this study. See Table 1 for sampling site codes as well as for further details for each population J Zoolog Syst Evol Res (2013) 51(2), 85--99 © 2013 Blackwell Verlag GmbH Table 1. List of samples sequenced, including populations codes, sampling localities, country, coordinates, collection data, number of individuals (N), substrates, some important description of the populations in diversity Species and GenBank accession numbers for COI and 18S sequences

Site, Locality, GenBank accession Species Code Country Coordinates Collection data N Substrate Notes nos. for COI; 18S Clade

Caprellaandreae CAALB Alboran, 35°56′26.35″N, 3°1′23.91″W 2001 8 Caretta caretta KC146127–KC146134 IIA Mayer, 1890 Alboran Sea, Spain arlapenantis Caprella CAAZO S~ao Miguel Island, 37°48′28″N, 23°33′80″W 2007 14 Drifting stick KC146135–KC146148; IIA Azores, Portugal JX988590–JX988592 CACHA Charleston, USA 28°23′14.64″N, 80°32′38.76″W 4 November 2006 4 Caretta caretta KC146149–KC146152; IIB JX988593–JX988595 CACOL Columbrestes, 39°51′19.2″N, 0°40′04.2″E 19 July 1996 2 Drifting material KC146153–KC146154 IIA Castellon, Spain CAFUE Fuengirola, 36°33′14.37″N, 4°29′21.39″ W 2001 6 Caretta caretta KC146155–KC146160 IIA

Malaga, Spain complex CAMOR El Morche, 36°44′15″N, 3°59′38″W 2 July 2011 15 Drifting rope KC146161–KC146175; IIA Malaga, Spain JX988596–JX988598 CATUR Seferihisar, 38°11′32″N, 26°38′07″E 1 April 2010 3 Caretta caretta KC146176–KC146178 IIB Izmir, Turkey Caprella dilatata CDALG Algeciras Bay, 36°10′0.5″N, 5°24′36.82″W September 2004 4 On buoys (algae and Without proximal projection on KC146180–KC146183; III Kroyer, 1843 Cadiz, Spain briozoans) male gnathopod 2 propodus KC146309 CDALI Guardamar, 38°05′1.10″N, 0°38′1.59″W 5 December 2006 4 Fish farm KC146184–KC146187; III Alicante, Spain JX988602, KC14631 CDCAT Itapocoroi Bay, 26°58′33″S, 48°35′51″W 19 October 2006 4 Mussel farm KC146179, III municipality of KC146188–KC146190 Penha, Santa Catarina, Brazil CDPAR Praia de Caoba, 26°51′32.54″S, 48°32′0.51″W 7 October 2006 4 Intertidal alga KC146191–KC146194 III municipality of Pterocladia Matinhos, Parana, capillacea Brazil CDPOR El Portil, 37°12′43″N, 7°4′26.80″W 11 June 2004 4 On buoys (algae and Without proximal projection on KC146195–KC146198 III Huelva, Spain briozoans) male gnathopod 2 propodus CDPORB El Portil, 37°12′43″N, 7°4′26.80″W21–22 May 2008 4 Dyctiota dichotoma KC146199–KC146202 III Huelva, Spain

olgSs vlRs(2013) Res Evol Syst Zoolog J Caprella penantis CPALM Punta Almina, 35°54′2.52″N, 5°16′29.68″W 16 August 2000 4 Intertidal alga Gelidium Without proximal projection on KC146203–KC146206 VA Leach, 1814 Ceuta, Spain sesquipedale male gnathopod 2 propodus CPAZU Playa Azul, 39°07′08.5″N, 9°23′35.3″W 5 August 2008 3 Intertidal alga Without proximal projection on KC146207–KC146209 VA Portugal Asparagopsis armata male gnathopod 2 propodus CPBAL Baleo, Spain 43°38′46.4″N, 8°06′17.4″W 2 August 2008 4 Intertidal alga With proximal projection on KC146210–KC146213; VB © Pterocladia capillacea male gnathopod 2 propodus KC146347 03BakelVra GmbH Verlag Blackwell 2013 CPBEN Benzu, Ceuta, 35°54′55.79″N, 5°22′26.81″W 19 June 2006 4 Intertidal alga Without proximal projection on KC146214–KC146217; VA Spain Asparagopsis armata male gnathopod 2 propodus KC146311 and Cystoseira tamarisciplia CPCAR Punta Carnero, 36°04′39.19″N, 5°25′20.71″W 23 June 2006 3 Intertidal alga Without proximal projection on KC146218–KC146220 VA Algeciras Bay, Asparagopsis armata male gnathopod 2 propodus Spain

51 CPCAST Castelejo, Portugal 37°06′9.3″N, 8°56′46.7″W October 2006 4 Intertidal alga With proximal projection on KC146221–KC146224; VA

2,85--99 (2), Pterocladia sp. and male gnathopod 2 propodus JX988601, KC14631 Gelidium sp. CPCET Cetarea, Spain 43°33′36.9.’N, 6°23′46.7′W 1 August 2008 3 Intertidal alga Cystoseira With proximal projection on KC146225–KC146227; VB

tamarisciplia male gnathopod 2 propodus KC146348 87 © olgSs vlRs(2013) Res Evol Syst Zoolog J Table 1. (continued) 88 03BakelVra GmbH Verlag Blackwell 2013

Site, Locality, GenBank accession Species Code Country Coordinates Collection data N Substrate Notes nos. for COI; 18S Clade

CPCEU Calamocarro, Ceuta, 35°53′59.31″N, 5°20′35.72″W 31 August 2004 2 Intertidal alga Without proximal projection on KC146228–KC146229 VA Spain Asparagopsis armata male gnathopod 2 propodus ′ CPCHO El Chorrillo, Ceuta, 35°52′26.95″N, 5°20 21.87′W 19 June 2006 3 Intertidal alga Without proximal projection on KC146230–KC146232 VA Spain Asparagopsis armata male gnathopod 2propodus CPEST Estepona, Malaga, 36°25′28.79″N, 5° 8′41.51″W 24 June 2006 3 Intertidal alga Corallina With proximal projection on KC146233–KC146235; VA Spain elongata male gnathopod 2 propodus JX988600 CPGIB Coquimbo, Chile 29°58′21.60″S, 71°21′21.06″W 30 October 2006 8 Floating buoys Caprella ‘verrucosa’ in KC146236–KC146243; I

51 Guerra-Garcia & Thiel 2001 KC146313–KC14632 2,85--99 (2), CPGUA Guadalmesõ, Cadiz, 36°01′54.69″N, 53°1′54.32″W 20 July 2006 1 Intertidal alga Corallina Without proximal projection on KC146244 VA Spain elongata male gnathopod 2 propodus CPHER Grottes d’Hercule, 35°46′9.31″N, 5°56′8.34″W 23 June 2006 3 Intertidal alga Cystoseira With proximal projection on KC146245–KC146247; VA Morocco tamarisciplia male gnathopod 2 propodus KC146349 CPISA Isla Isabel-Las 21°51′21″N, 105°52′42″W 29 January 2008 4 Leptogorgia cf. rigida Without ‘grasping spines’ KC146248–KC146251; VIA Monas, Mexico (6m) KC146321–KC14632 CPJAP Kami-Amakusa, 32°31′00″N, 103°26′00″E 13 September 2010 10 Bugula neritina With proximal projection on KC146252–KC146261; IV Kumamoto, Japan male gnathopod 2 propodus KC146325–KC14633 ′ CPKSAR ksar-es seguir, 35°50 40.88″N, 5°33′28.18″W 23 June 2006 3 Intertidal alga Asparagopsis Without proximal projection on KC146262–KC146264; VA Morocco armata and Gelidium male gnathopod 2 propodus KC146350 sesquipedale CPLAB Labruge, Portugal 41°16′47.05″N, 8°43′52.7″W 4 August 2008 3 Intertidal alga With proximal projection on KC146265–KC146267; VA Corallina elongata male gnathopod 2 propodus KC146335 CPMIN Mindelo, Portugal 41°20′13.02″N, 8°44′43.67″W October 2006 4 Intertidal alga With proximal projection on KC146268–KC146271; VA Cystoseira sp. male gnathopod 2 propodus KC146336 CPOGE Ogella, Spain 43°22′27.0″N, 2°32′25.8″W 30 July 2008 1 Intertidal alga With proximal projection on KC146272 VB Corallina elongata male gnathopod 2 propodus ′ CPOYA Oyambre, Spain 43°24′14.2″N, 4°20 59.00″W 31 July 2008 1 Intertidal alga With proximal projection on KC146273 VB Asparagopsis armata male gnathopod 2 propodus CPOYAB Oyambre, Spain 43°24′14.2″N, 4°20′59.00″W 31 July 2008 4 Intertidal alga Cystoseira With proximal projection on KC146274–KC146277 VB C

tamarisciplia male gnathopod 2 propodus ABEZAS ′ CPOYAS Oyambre, Spain 43°24′14.2″N, 4°20 59.00″W 31 July 2008 4 Submareal alga Gelidium With proximal projection on KC146278–KC146281; VB sesquipedale male gnathopod 2 propodus KC146337–KC14633

CPPAJ Isla Pajaros, 23°15′06″N, 106°28′24″W 29 January 2008 4 Hydroids on gorgonians Without ‘grasping spines’ KC146282–KC146285; VIB ,C – Mexico (Leptogorgia spp.) KC146339 KC14634 ABEZAS CPPTO Puerto de Ceuta, 35°53′38.11″N, 5°19′10.36″W 1 August 2004 4 Intertidal alga Without proximal projection on KC146286–KC146289 VA Ceuta, Spain Asparagopsis armata male gnathopod 2 propodus

CPSAF Saﬁ, Morocco 32°18′36.77″N, 9°15′00″W 21 June 2006 2 Intertidal alga With proximal projection on KC146290–KC146291; Vc ,M –

Gelidium sp. male gnathopod 2 propodus, KC146343 KC14634 ACHORDOM but it’s something different to the others specimens CPTAR Tarifa Island, 36°00′00.7″N, 5°3637.5″W 5 August 2004 4 Intertidal alga Asparagopsis Without proximal projection on KC146292–KC146295; VA Cádiz, Spain armata male gnathopod 2 propodus JX988599, KC14634 CPTARB Tarifa Island, 36°00′00.7″N, 5°3637.5″W 5 August 2004 4 Intertidal alga Gelidium Without proximal projection on KC146296–KC146299 VA G and Cádiz, Spain sesquipedale male gnathopod 2 propodus ° ′ ″ ° ′ ″ – CPTOR Torreguadiaro, 36 18 01.2 N, 5 15 55.5 W 1 October 2006 4 Intertidal alga Gelidium With proximal projection on KC146300 KC146303; VA UERRA Cadiz, Spain spp. male gnathopod 2 propodus KC146346 CPTORB Torreguadiaro, 36°18′01.2″N, 5°15′55.5″W 4 December 2005 1 Intertidal alga Asparagopsis With proximal projection on KC146304 VA Cadiz, Spain armata male gnathopod 2 propodus -G ARCÍA CPTPO Tarifa east Island, 36°00′17.48″N, 5°36′41.41″W 4 October 2008 3 Intertidal alga Asparagopsis With proximal projection on KC146305–KC146307 VA Cádiz, Spain armata male gnathopod 2 propodus Species diversity in Caprella penantis complex 89

Table 2. Ampliﬁcation primers pairs used for COI ampliﬁcation in this study

Primer Sequence (5′-3′)Ta Source

Universal First fragment LCO1490 GGT CAA CAA ATC ATA AAG ATA TTG G 45.0 Folmer et al. (1994) HCO2198 TAA ACT TCA GGG TGA CCA AAA AAT CA Folmer et al. (1994) Second fragment COI2F TTY GAY CCI DYI GGR GGA GGA GAT CC 42.7 Otto and Wilson (2001) COI2R GGR TAT TCW GAR TAW CGN CGW GGT AT Otto and Wilson (2001) Caprella First fragment Cp_COIF1 TTA AGA ATA ATT ATT CGT ACA G 47.2 This study Cp_COIF2 GGA GAT GAY CAA ATT TAT AAT G 45.0 This study Cp_COIR1 AAT ATA YAC TTC TGG RTG ACC This study Second fragment Cp_COIF3 GGT CAY CCA GAA GTR TAT ATT 47.2 This study Caprella penantis First fragment penanF1 GTR GTA ACA GCA CAC GCC TT 52.5 This study penanR640 GCC CCA AAA GTT TCT TTC TT This study Second fragment penan 579 CCT GCC TTY GGT ATT GTC TC 50.5 This study penanR1140 CCT CTT AAA CCT MRT ATA TGY TG This study

Ta, annealing temperature. primers, PCR amplifications were carried out in a 50-ll reaction volume ysis using Gblocks (Castresana 2000). Uncorrected p-distances between consisting of 3 ll(~3–25 ng) template DNA, 5 ll of the corresponding taxa were also calculated using MEGA version 5.05 (Tamura et al. 109 buffer MgCl2 free (Biotools, Madrid, Spain), 3 ll MgCl2 (50 mM), 2011). 1 ll of dNTPs mix (10 mM), 0.8 ll of each primer (10 mM), 0.5 ll Bovine Serum Albumin (BSA Acetylated, 10 mg mlÀ1; Promega, Madrid, Spain), 0.3 ll Taq DNA polymerase (5 U llÀ1; Biotools, Phylogenetic analyses Madrid, Spain) and double-distilled water (ddH O). PCR amplifications 2 Phylogenetic analyses were performed using three different methods to were performed under the following conditions: initial 4-min denaturation verify whether alternative topologies were supported by different tree- at 94°C, followed by 40 cycles of 45 s at 94°C, 1 min at 42–53°C (opti- building approaches: maximum parsimony (MP), maximum likelihood mized for each primer pair; see Table 2) and 1 min at 72°C. The amplifi- (ML) and Bayesian Inference. Only one individual (or sequence) per vari- cation ended with a final extension step at 72°C for 10 min. ant haplotype was included. Unfortunately, trees for the COI and 18S 18S fi In addition, a 1051-bp fragment of the nuclear gene was ampli ed genes could not be rooted using the same species as outgroups (see by the primers 18S-ai and 18S-bi described by Whiting (2002) in a sub- below). Therefore, both data sets were analysed separately. set of 56 representative individuals of each clade (see results; Figs 2 and To test the occurrence of saturation in the COI gene, uncorrected 3 and Table 1). PCR amplifications were achieved using 2 mM of MgCl 2 p-distance values between haplotypes were plotted against ML dis- under the following conditions: initial 4-min denaturation at 94°C, fol- tance. ML distances were obtained using the parameters of the evolu- lowed by 35 cycles of 45 s at 94°C, 45 s at 56–60°C and 1 min at 72°C. tionary model coestimated with the ML tree. Final extension was achieved at 72°C for 12 min. The MP analysis was performed using MEGA version 5.05 (Tamura The PCR products were visualized under blue light on 1% agarose gel et al. 2011) using 1000 bootstraps analyses to estimate branch support. stained with SYBR Safe (Invitrogen, Madrid, Spain), with a comigrating The ML analysis was run in PHYML (Guindon et al. 2010) with the most 100-bp ladder molecular-weight marker to confirm their correct amplifica- appropriate best-fit substitution model of DNA evolution determined with tion. The amplified fragments were purified by ethanol precipitation prior the Akaike information criterion (Akaike 1974) implemented in JMODEL- to sequencing both strands by Secugen S.L. (Madrid, Spain) using the ® TEST 0.1 (Posada 2008). The best-fit models selected were TIM2 + I + G BigDye Terminator v3.1 (Applied Biosystems, ABI, Madrid, Spain). and TVMef + I + G, for the COI and 18S data sets respectively. Because Sequences were checked and edited using Sequencher program (Gene of the limited number of models included in the available software, the Codes Corporation, Ann Arbor, MI, USA). They were verified as Caprel- GTR + I + G model of evolution was used. Branch support was esti- lidea DNA using GenBank BLASTn searches (Altschul et al. 1990) and mated using 1000 bootstraps. The Bayesian phylogenetic inference analy- were thereafter deposited in GenBank (Accession nos: JX988590- sis was performed using the program MRBAYES version 3.1.2 (Ronquist JX988602, KC146127-KC146350, Table 1). and Huelsenbeck 2003), implementing the GTR + I + G model. A four- chain metropolis-coupled Markov chain Monte Carlo (MCMCMC) analy- 7 Estimates of genetic diversity sis was run twice in parallel for 3 9 10 generations, and trees and parameters were sampled every 1000 generations with the heating param- For COI gene, all sequences were aligned using CLUSTALW (Thompson eter set to 0.1. A majority-rule consensus tree was estimated combining et al. 1994) implemented in BioEdit (Hall 1999). Furthermore, sequences results from duplicated analyses, after discarding the first 3000 samples were uploaded in DNASP (Librado and Rozas 2009) and translated into as burn-in (corresponding to 10% of the total samples). Adequate burn-in amino acids to search for stop codons that would be indicative of the was tested using the software TRACER version 1.5 (Rambaut and Drum- presence of pseudogenes. mond 2007). Uncorrected p-distances were calculated using MEGA version 5.05 The phylogenetic relationships between Caprellidea are poorly under- (Tamura et al. 2011) and were used to estimate genetic divergence stood, making the selection of an appropriate outgroup difficult. Recent between pairs of taxa. Estimates of haplotype (Hd) and nucleotide diversi- molecular analyses carried out by Ito et al. (2011) based on the 18S gene ties (p) were calculated for all C. penantis’ clades found (see results; have indicated that a close relationship between Cyamidae (another fam- whenever the number of sampled individuals was more than four), using ily belonging to suborder Caprellidea) and Caprellidae exists. Thus, the ARLEQUIN version 3.5 (Excoffier and Lischer 2010). COI gene sequence from one Cyamidae species, Cyamus ovalis Roussel Sequences of 18S were aligned using the MAFFT algorithm (Katoh de Vauzeme, 1834, obtained from GenBank (accession no. DQ094901), et al. 2005), and highly variable regions were eliminated from the anal- was selected as outgroup. On the other hand, for the 18S, trees were J Zoolog Syst Evol Res (2013) 51(2), 85--99 © 2013 Blackwell Verlag GmbH 90 CABEZAS,CABEZAS,MACHORDOM and GUERRA-GARCÍA

Fig. 2. Bayesian consensus tree obtained for the mtDNA COI gene. The tree was rooted with Cyamus ovalis sequence available in GenBank. Values of nodes correspond to Bayesian posterior probabilities and bootstrap support given by the maximum likelihood analysis (both above branches), and to bootstrap support given by the maximum parsimony analysis (below branches) respectively (‘–’ indicates <50% support). Asterisks indicate the presence of proximal projection in adult male gnathopod 2 propodus rooted using two outgroups from the same genus (Caprella equilibra C. andreae haplotypes. This clade was further subdivided into Say, 1818 and Caprella danilevskii Czerniavskii, 1868) with sequences two well-supported monophyletic and geographically distinct available in GenBank (accession numbers: AY743950 and AB295398 clades: Clade IIA, composed by all haplotypes from eastern respectively). Atlantic and western Mediterranean (CAAZO, CAMOR, CA- Haplotype genealogy was also investigated for the COI gene by build- COL, CAALB and CAFUE; see Fig. 1 and Table 1); and Clade ing a network of haplotypes using TCS version 1.21 (Clement et al. II , which included all C. andreae haplotypes from western 2000) with a 90% statistical parsimony connection limit. B Atlantic and eastern Mediterranean (Charleston and Turkey; Fig. 2). Clade III included all C. dilatata haplotypes, but did not Results show further subdivision despite the high geographical distance between sampled locations. Clades II and III appeared as sister Phylogenetic inference group in the analyses only when using the information from the Phylogenetic analyses of the COI data set using the three differ- mitochondrial COI gene, although it displayed little or no sup- ent approaches rendered phylogenetic trees with similar overall port in the ML or the MP analyses respectively (Fig. 2). Clade topologies, with main clades receiving high bootstrap or posterior IV included all C. penantis haplotypes from Japan, which was probabilities support (>75% and >0.99% respectively). All analy- sister to the clade composed of Clades II and III. Clade V con- ses revealed six main well-supported clades, indicated as I–VI in tained all C. penantis haplotypes from the Iberian Peninsula and Fig. 2. Clade I was the most basal one and included all C. pe- Morocco. Within this clade (corresponding to the great majority nantis haplotypes from Coquimbo, Chile. Clade II grouped all of C. penantis specimens), evidence of geographical structure of J Zoolog Syst Evol Res (2013) 51(2), 85--99 © 2013 Blackwell Verlag GmbH Species diversity in Caprella penantis complex 91 genetic variation was observed. It split into three highly sup- mens with proximal projection on gnathopod 2 (Gn2) propodus, ported clades: Clade VA that grouped all haplotypes from the C. penantis f. testudo and lusitanica, indicated by an asterisk in Strait of Gibraltar and Portugal; Clade VB composed of all hapl- the tree in Fig. 2. otypes from northern Spain; and Clade IVC, where only C. pe- The reconstruction of the haplotype network for all C. penan- nantis haplotypes from Safi (Atlantic Morocco) were included. tis sequenced data retrieved seven separate networks that could Finally, Clade VI grouped all C. penantis haplotypes from both not be connected using the 90% parsimony connection limit Mexican locations. Further subdivision occurred within the Mexi- (Fig. 4). Not unexpectedly, they coincided with the C. penantis can clade where two highly supported clades containing haplo- clades observed in the phylogenetic analyses (see Figs 2 and 3). types from Isla Isabel (CPISA) and Isla Pajaros (CPPAJ) were All networks showed a marked genetic structure with a large recovered (Fig. 2). number of haplotypes per locality and separated by a large num- Although the 927 bp of the nuclear gene 18S also confirm the ber of mutational steps (Fig. 4). A total of 79 haplotypes was existence of these divergent clades, they had weak support in all observed for the 105 individuals considered (Table 3). Within analyses and relationships between them are not clear (Fig. 3). individuals belonging to Clade VA, corresponding to the great Unlike the COI gene, Clade II was estimated to be paraphyletic, majority of C. penantis sampled localities, a total of 41 haplo- and Clades II and III did not appear as sister group in all the types were observed for the 60 individuals considered (see analyses conducted (Fig. 3). Table 3). Only three haplotypes were shared between two or Overall, C. penantis was found to be polyphyletic in all analy- three localities and no central haplotype could be distinguished ses, for all data sets (COI and 18S, Figs 2 and 3 respectively). by higher frequencies. The remaining haplotypes were private to Interestingly, phylogenetic trees did not group together speci- a specific locality. Differences between haplotypes from different

Fig. 3. Bayesian consensus tree obtained for the 18S gene. The tree was rooted with Caprella equilibra and Caprella danilevskii sequences available in GenBank. Node values correspond to Bayesian posterior probabilities and bootstrap support given by the maximum likelihood analysis (both above branches), and to bootstrap support given by the maximum parsimony analysis (below branches) respectively (‘–’ indicates <50% support) J Zoolog Syst Evol Res (2013) 51(2), 85--99 © 2013 Blackwell Verlag GmbH 92 CABEZAS,CABEZAS,MACHORDOM and GUERRA-GARCÍA

(a) (b) (c)

(d) (e)

(f) (g)

Fig. 4. Mitochondrial (COI) haplotypes network (90% parsimony connection limit) of Caprella penantis. Haplotype networks (A) and (B) correspond to haplotype relationships between individuals from Coquimbo (Chile) and Japan respectively (Clades I and IV in the phylogenetic tree; Fig.2). Haplo- type networks (C), (D) and (E) represent the relationships between individuals from Iberian Peninsula and Morocco (corresponding to Clades VA,VB and VC, respectively, in the phylogenetic tree; Fig. 2). Haplotype networks (F) and (G) correspond to haplotype relationships between individuals from Mexico (Clade VIA and VIB, respectively, in the phylogenetic tree). Circle sizes are proportional to the number of haplotypes, and localities are coded by ﬁlling patterns. Non-observed haplotypes are represented by small white circles. Each line connecting haplotypes represents a single mutational change, as well as the number on the white boxes sampled localities were on the order of 1–58 mutations C. andreae and C. dilatata respectively. Within C. penantis, the (Fig. 4C). Within this network, a star-like phylogeny was recov- highest values of haplotype diversity were found in C. penantis ered for Ceuta’s localities (CEU, ALM, PTO, BEN and CHO). from Isla Isabel (Clade VIA) and C. penantis from the Strait of Gibraltar and Portugal (Clade VA), and the lowest in C. penantis from Isla Pajaros (Clade V ). Nucleotide diversity ranged from Genetic diversity B 0.0012 in Clade VIB to 0.00121 in C. penantis from Japan The COI sequence data set obtained for C. penantis and the clos- (Clade IV; Table 3). est species’ individuals sequenced, after alignment, consisted of For COI, genetic distances among the different clades belong- 1137 characters, of which 694 were constant and 443 were vari- ing to C. penantis (Clades I, IV, V and VI) ranged from 11.7% able, of which 420 were parsimony informative including the to 16.5%. Individuals from Coquimbo (Clade I), Japan (Clade outgroup species. IV) and Mexico (Clade VI) were 14–14.9%, 11.7–12.3% and For the mtDNA COI gene, haplotype and nucleotide diversi- 13.5–14.8%, respectively, divergent from individuals of the main ties were generally high (Table 3). A mean haplotype diversity C. penantis clade (Clade V). Within Clade V, uncorrected p-dis- of 0.978, 0.869 and 0.781, and nucleotide diversity of 0.1018, tance among the distinct subclades was in the range 6.3–7.1%. 0.0246 and 0.0092 were obtained for C. penantis complex, Divergence between C. andreae (Clade II) and C. dilatata J Zoolog Syst Evol Res (2013) 51(2), 85--99 © 2013 Blackwell Verlag GmbH Species diversity in Caprella penantis complex 93

Table 3. Estimates of genetic diversity for genetic clusters of Caprella showed bigger gills and were more robust and larger than the found based on mitochondrial COI sequence other individuals belonging to the forms testudo or lusitanica (Fig. 5C). Individuals from Japan also looked similar to the p Æ Clade NH Hd ( SD) forms testudo or lusitanica, but had both more robust and bigger I 8 6 0.929 0.0070 (0.0012) Gn2 and antenna 1 (Fig. 5D). The presence of abundant dorsal II tubercles on pereonites 5, 6 and 7, as well as the robustness of IIA 45 14 0.829 0.0092 (0.0048) antenna 1, indicate that individuals from Coquimbo, Chile, IIB 7 4 0.809 0.0085 (0.0051) clearly belong to the endemic C. penantis f. gibbosa described III 24 14 0.781 0.0092 (0.0048) from Coquimbo by Mayer (1890, 1903). Guerra-García et al. IV 10 9 0.956 0.0121 (0.0067) (2006) called this form ‘verrucosa’ based on the tubercles, but V this is a different species from Caprella verrucosa (Fig. 5E). On VA 60 41 0.983 0.0120 (0.0061) VB 17 15 0.978 0.0057 (0.0032) the other hand, the P5-7 propodi were clearly elongate and VC 22––‘grasping spines’ were lacking in specimens collected from gor- VI gonians on Mexico (Fig. 5F). VIA 4 4 1 0.0022 (0.0018) Caprella dilatata could be morphologically distinguished from VIB 4 2 0.667 0.0012 (0.0011) C. penantis mainly by the body size (individuals of C. dilatata were usually larger than those belonging to C. penantis), the gill N, number of individuals; H, number of haplotypes; Hd, haplotype diversity; p, nucleotide diversity; SD, standard deviation. shape (elongate in C. penantis and round in C. dilatata) and the Gn2 (presence of a median pointed tooth and subdistally trapezoid process in C. dilatata and absence in C. penantis; Fig. 5G). On the other hand, C. penantis differed from C. andreae mainly (Clade III) was in the range 7.6–9.0%. Divergence between these by having concave P5-7 propodi palm (Fig. 5H). caprellid species relative to all C. penantis clades were in the range 9.1–15.4% (Table 4). The 18S alignment was 927-bp long, and included a total of Discussion 65 variable sites, of which 37 were parsimony informative. A Taxonomic and ecological remarks total of 13 haplotypes were observed for the 56 individuals considered. For the 18S gene, genetic divergence among C. penantis The family Caprellidae is one of the most problematic groups clades was in the range 0.7–2.5%. Divergence between Clade I, among Crustacea, and Amphipoda in particular, concerning their IV and VI relative to Clade V were 1.9–2.5%, 0.8–1.5% and 1.6 taxonomy, mainly due to their high degree of intraspecific mor- –1.9% respectively. Within Clade V, genetic divergence among phological variability. Our results show that the mitochondrial distinct clades ranged from 0.2% to 0.5%. Divergence between COI gene is useful for clarifying systematic questions in the C. andreae and C. dilatata was 1.0%. Divergence between these main genus Caprella. Moreover, despite being a highly con- species and C. penantis ranged from 0.3–2% to 0.8–1.8% respec- served gene in caprellids, 18S proved to have also a good dis- tively (Table 4). Interspecific divergence between C. equilibra criminatory power for identifying species within this genus. The and C. danilevskii was 3.7%, whereas of C. equilibra and C. da- large genetic divergence (7.6–15.4% for COI, and 1–1.9% for nilevskii relative to C. penantis was 3.8–4.5% and 1.2–1.9% the 18S gene) between C. penantis, C. dilatata and C. andreae, respectively. comparable to those reported for other peracarid crustaceans (Ashton et al. 2008; Hou and Li 2010; Baird et al. 2011), strongly support these taxa as separate and good species, tradi- Morphological data tionally considered altogether under the ‘acutifrons’ complex We found some slight morphological differences among speci- (Mayer 1890, 1903). These results also confirm that morphologi- mens from different C. penantis populations (see Table 1 for cal characters such as gill shape, Gn2 shape and concavity/con- more details and Fig. 5). Individuals collected from Iberian Pen- vexity of P5-7 propodi palm (Fig. 5) are indeed useful for insula and Morocco basically matched with the form simulatrix delineating and identifying individuals to species in the genus (without proximal projection on the propodus of male Gn2; Caprella. They also support the results obtained in the only two Fig. 5A1) or lusitanica and testudo (with this projection; previous molecular studies, both of them based on RAPD tech- Fig. 5A2). Specimens from localities of the North of Spain nique (Guerra-García et al. 2006; Cabezas et al. 2010). (Ogella, Oyambre, Cetarea, Baleo, Fig. 1) also depicted a den- Interestingly, within C. penantis sensu stricto (Clade V), phy- sely setose and bigger Gn2 propodus palm (Fig. 5B); and those logenetic trees did not show a clear separation between the popu- from Safi, Morocco, showed the proximal projection on Gn2, but lations with proximal projection on the propodus of male Gn2

Table 4. Percentage of average sequence divergence values (based on uncorrected p-distances) between major mitochondrial COI (lower diagonal) and nuclear 18S (upper diagonal highlighted) clades identiﬁed by phylogenetic analyses

Clade I Clade IIA Clade IIB Clade III Clade IV Clade VA Clade VB Clade VC Clade VIA Clade VIB

Clade I – 2.0 1.9 1.8 2.0 1.9 2.0 2.5 2.0 2.0 Clade IIA 14.9 – 1.4 1.0 0.3 1.0 1.2 1.0 1.9 1.9 Clade IIB 15.4 7.7 – 1.0 1.1 1.2 1.4 1.4 1.6 1.6 Clade III 14.2 7.6 9.0 – 0.8 0.9 1.2 1.4 1.4 1.4 Clade IV 15.8 11.2 11.6 11.1 – 0.7 0.8 0.9 1.5 1.5 Clade VA 14.0 10.0 10.3 9.1 12.1 – 0.2 0.4 1.6 1.6 Clade VB 14.9 10.2 10.2 10.2 11.7 7.1 – 0.5 1.7 1.7 Clade VC 14.0 9.3 9.6 9.2 12.3 6.3 7.1 – 1.9 1.9 Clade VIA 16.3 14.7 15.4 14.1 15.6 13.9 14.8 14.0 – 0.2 Clade VIB 16.5 14.2 14.0 14.0 14.6 14.2 14.3 13.5 5.3 –

(a) (b)

(e) (f)

(g) (h)

Fig. 5. Adult males’ photographs of the different Caprella species included in this study, with indication (white arrow) of the main diagnostic characters. (A1) Caprella penantis form simulatrix, characterized by the presence of a triangular projection on the cephalon, rounded gills, ‘grasping spines’ on the pereopods 5–7 propodi (see smaller picture) and by the absence of proximal projection on gnathopod 2 (Gn2) propodus. (A2) C. penantis form testudo/lusitanica, which differs of the previous form only in the presence of proximal projection on Gn2 propodus. (B) C. penantis from the north of Spain, showing a quite setose and bigger Gn2 propodus palm. (C) C. penantis from Saﬁ (Morocco), with bigger gills and more robust and larger body. (D) C. penantis from Japan, showing more robust and bigger Gn2 and antenna 1. (E) C. penantis f. gibbosa, characterized by the presence of dorsal tubercles on pereonites 5–7 and robustness of antenna 1. (F) C. penantis from Mexico, with pereopods’ propodus elongate and without ‘grasping spines’ (see the smaller photograph). (G) Caprella dilatata, note presence of a median tooth and subdistally trapezoid process on Gn2 and elongate gills. (H) Caprella andreae, showing concave pereopods 5–7 palm

(forms testudo and lusitanica; indicated by an asterisk) with Variation in the ability of attached organisms to respond physi- respect to populations without this projection (simulatrix; see cally to different habitats can condition individual survivorship Fig. 2). The presence of this projection has long been considered and result in morphological variability. Similar results have been as a useful taxonomic character in C. penantis. Nevertheless, reported for other caprellid species through experimental evi- according to these results, it is not an informative character and dence by Aoki and Kikuchi (1990). They found that body spin- different forms (testudo/lusitanica and simulatrix)ofC. penantis ation may not be a useful character for separating species, and may correspond to intraspecific variation. The higher morpholog- that the intraspecific morphological variation is the result of habi- ical variability detected within C. penantis could be a result of tat variation, such as differences of temperature, habitat condi- their distinct habitat preferences (Bynum 1980; Caine 1989; Aoki tion, food availability and interspecific interactions. In the case and Kikuchi 1995) as this species has been recorded from a wide of C. penantis, both morphological forms were found within the variety of substrata, for example, algae, gorgonians, hydroids, same locality. The existence of different conditions related with etc. (McCain 1968; Laubitz 1972; Díaz et al. 2005). Some struc- hydrodynamism, concentration of dissolved oxygen as well as tural habitats may impose stricter functional constraints than oth- differences in wind exposure, could explain the presence of dif- ers, leading to stronger selection resisting diversification away ferent morphological forms of C. penantis. Bynum (1980) did a from morphologies that confer effective use of that surface. morphometric study on caprellids from coastal and estuarine sites J Zoolog Syst Evol Res (2013) 51(2), 85--99 © 2013 Blackwell Verlag GmbH Species diversity in Caprella penantis complex 95 in North Carolina and found a gradient of forms in that body of two zoogeographical zones, which are separated by a relatively parts and appendages associated with grasping depending on the wide transitional zone (between 30°S and c. 45°S; Thiel 2002), degree of exposure to turbulence (‘ecotopic variation’; Caine could promote the speciation and the subsequent endemism of all 1989; Guerra-García 2001). On the contrary, despite the great these species. The results presented here could be another exam- geographical distance between some of the C. andreae (Clade II) ple of their considerable morphological differences found within and C. dilatata (Clade III) populations analysed, they both show C. penantis f. gibbosa with respect to C. penantis sensu stricto, low levels of intraspecific variability. These species have more supporting that this form could also merit specific rank. On the specific habitat preferences living mainly associated with drifting other hand, the absence of ‘grasping spines’ in Mexican popula- objects such as buoys, ropes, pieces of wood, and even C. an- tions has been reported by McCain (1968) in samples of C. pe- dreae has also been reported clinging to the algal substrate on nantis taken on Leptogorgia. Their adaptation to ecologically turtle carapaces (Krapp-Schickel 1993; Sezgin et al. 2009; Zak- isolated habitats, such as gorgonians, could have led to their spe- hama-Sraieb et al. 2010). According to Aoki and Kikuchi ciation, as was remarked by Aoki and Kikuchi (1995) for C. an- (1995), C. andreae may have diversified from the old ‘acuti- dreae. So, the presence/absence of ‘grasping spines’ could be frons’ complex by adapting to ecologically isolated habitats such considered as a useful taxonomic character in the genus Caprella. as turtle carapaces. Mexican samples were collected in two different islands (Isla Isa- bel and Isla Pajaros) approximately 164 km apart. These populations differ only in the length of pereopods (larger in specimens Cryptic speciation within C. penantis from Isla Pajaros) and body size (more robust in Isla Isabel). Our results showed a considerable sequence divergence (5.3% for Although species delimitation continues to be one of the most COI, see Table 4) at the level of congeneric interspecific differen- controversial issues in biological science (de Queiroz 2007), sev- tiation. Isolation is a characteristic feature of islands (Emerson eral criteria are now considered to provide a valid argument for 2002), promoting differentiation and speciation through the reduc- species boundaries based on genetic clades (see Baird et al. tion or even cessation of gene flow between insular populations 2011). According to our results, populations belonging to the and between insular and continental populations. The reciprocal supposedly cosmopolitan species, C. penantis, were separable monophyly of haplotypes typical of these two Mexican islands into four distinct monophyletic mitochondrial lineages (Clades I, suggests that there is no genetic exchange between them. IV, V and VI; Fig. 2) that correlate with their geographical dis- Although a more detailed analysis is needed to confirm the tributions and distances among those regions, satisfying the absence of gene flow, the described pattern can be taken as a requirements of the phylogenetic species concepts (Baum and strong indication that these could be two distinct species. Shaw 1995). Furthermore, given that divergence at COI gene On the other hand, our results also challenge the morphologi- exceeds that between those recognized as valid species (Hebert cal taxonomy of C. penantis by providing strong molecular evi- et al. 2003; Witt et al. 2006; Costa et al. 2007; Table 4) as well dence of cryptic speciation, concordant across mitochondrial and as the formation of unconnected haplotype networks in statistical nuclear markers (Figs 2 and 3). This is the case for specimens parsimony analyses (Fig. 4), we believe that they may represent from Japan (Clade IV). Although cryptic species delineation is a potentially new species (Witt and Hebert 2000; Hogg et al. more complex process in marine habitats due to the fact that spe- 2006; Hart and Sunday 2007). Reconstructions based on 18S ciation process may be less coupled to morphological characters marker presented low levels of genetic variation (maximum 2%) than to other non-visible traits (Knowlton 1993, 2000), this due to its more conservative nature (Barrowclough and Zink Clade also satisfies the proposed amphipod COI species screen- 2009), but were in concordance with reconstructions provided by ing threshold (Witt et al. 2006) as well as the ‘4x’ rule that has COI data set (see Fig. 3 and Table 4). Rafting on floating sub- been used as a conservative approach to delimit cryptic species strata has been suggested as the process permitting long-distance in crustaceans (Birky et al. 2005; Morrone et al. 2010). The phe- dispersal of brooding marine invertebrates (Castilla and Guinez~ notypic similarity of species genetically well differentiated, as 2000; Fraser et al. 2011; Hoeksema et al. 2012) and, in particu- that here studied (Fig. 5D), could be interpreted as the result of lar, caprellids (Thiel et al. 2003b; Thiel and Gutow 2005). How- recent speciation, but also may derive from speciation with lack ever, in this study, the high levels of genetic differentiation of phenotypic differentiation due to similar habitat conditions. among populations suggest limited dispersal among geographi- Rocha-Olivares et al. (2001) also observed that in organisms cally isolated populations. Isolation by distance leading to with small body size, the number of taxonomically relevant char- restricted gene flow could have promoted species diversification acters decreases rapidly after speciation events. This incongru- among isolated populations (France and Kocher 1996), but spa- ence between morphological and molecular markers has already tial heterogeneity of habitats may also play a role in allopatric been identified in amphipod studies focused on gammarids (Witt speciation (Rogers 2007; Baird et al. 2011). et al. 2006; Pilgrim and Darling 2010; Baird et al. 2011), and is At the same time, we found that C. penantis f. gibbosa from likely to be associated with a cryptic species complex. In their Coquimbo, Chile (Clade I) and populations from Mexico (Clade study focused on the penaeid shrimp, Palumbi and Benzei (1991) VI) could be morphologically distinguished by a combination of suggested that this incongruence could be the consequence of the some constant characters from C. penantis sensu stricto (Clade faster evolutionary rate of mtDNA, as well as the slower rate of V; Fig. 5E–F). In addition, we found that these species have allo- morphological divergence possible due to stabilizing selection on patric distributions: C. penantis f. gibbosa was only found in Co- morphological or ecological characters. Comparative molecular quimbo region attached to buoys, and populations from Mexico and ecological studies are necessary to assess how ecological were collected from gorgonians at 6-m depth (see Table 1). factors may restrict morphological diversification. Therefore, molecular, morphological and ecological data support all two C. penantis f. gibbosa and C. penantis from Mexico being new species. Interestingly, a relatively high number of per- Genetic structure within Caprella penantis sensu stricto acarid species appear to be endemic to Chile (Thiel 2002; Thiel (Clade V) et al. 2003a), such as the caprellid species Deutella venenosa Mayer, 1890 (Thiel et al. 2003b). The particular oceanographic Results from the present work revealed a strong pattern of conditions along the Pacific coast of Chile, that is, the existence genetic and geographical structure between C. penantis J Zoolog Syst Evol Res (2013) 51(2), 85--99 © 2013 Blackwell Verlag GmbH 96 CABEZAS,CABEZAS,MACHORDOM and GUERRA-GARCÍA populations from the Iberian Peninsula and Morocco (Clade V; cosmopolitan species are really species complexes with strong Figs 2 and 4). Northern Iberian Peninsula haplotypes formed a geographical structure (Remerie et al. 2006; Chen and Hares highly differentiated clade relative to those from Portugal and 2011). The results observed in this study could be another exam- Strait of Gibraltar localities (Clades VB and VA respectively), as ple, supporting strong evidence that C. penantis is a complex of well to the ones from North Atlantic Morocco coast (Safi; Clade at least four species, of which one is reported as a cryptic spe- VC). This high level of genetic structuring with almost no cies. The existence of cryptic species in the marine environment mtDNA haplotypes sharing between sites has been reported in seems to be a far more widespread phenomenon as previously several marine species (see Lejeusne and Chevaldonne 2006; thought (Knowlton 1993, 2000). However, this study is to our Remerie et al. 2006; Xavier et al. 2009, 2011) and has often knowledge the first in reporting evidence of cryptic speciation been attributed to the large population sizes of marine species within a caprellid species. Hence, the current species status (Bucklin and Wiebe 1998). Our results show two main genetic within the genus Caprella may still be an underestimation of the breaks along the study area: the first one separates the Northern actual species diversity of this genus. On the other hand, the Atlantic Moroccan coast from the Atlantic Iberia and the Medi- observed pattern of genetic and geographical structure between terranean Sea, whereas the second separates the Northern Atlan- populations of C. penantis from Iberian Peninsula and Morocco tic Iberia (Bay of Biscay) from the Southern Atlantic. A similar suggest that not only restricted dispersal but also historical isola- pattern in the same geographical region has been observed in tion occurred in the past history of this species. Clearly, further another peracarid species: Stenosoma nadejda (Rezig, 1989), investigation involving a larger set of individuals from a more where the genetic break separating the Northern portion of the complete geographical sampling using a larger set of molecular Atlantic Moroccan coast from the Atlantic Iberia and the Alboran markers must be carried out to resolve the identity, evolutionary Sea was detected (Xavier et al. 2009, 2011). The second has origin and diversification of the C. penantis complex, as well as never been reported in the literature. Studies focused in this for a better understanding of the patterns of genetic connectivity region failed to detect any significant break (Patella rustica observed. Linnaeus, 1758: Ribeiro et al. 2010; and Pollicipes pollicipes (Gmelin, 1790; Campo et al. 2010), which is not surprising given their potential for dispersal during the larval stage. On the Acknowledgements other hand, the existence of particular hydrographic features in Special thanks to F. Espinosa, R. Xavier, A. Ito, R. King, M. Thiel, S. the Portuguese coast, with a cooler northern region affected by Masunari, J.E. Sanchez-Moyano, M. Sezgin and the Centro de Recupe- upwelling, and a much warmer south region with a strong Medi- racion de Especies Marinas Amenazadas (Centre for the protection of terranean influence (Santos et al. 2004) could both explain the endangered marine species) (CREMA; Malaga) for providing some of the high differentiation of the northern Spain samples and the higher samples used in this study. We are very grateful to Pilar Ochoa and Isa- similarity of all Portugal samples with those from the Strait of bel Bermudez de Castro (National Museum of Natural Science, Madrid) for their kind help, support and assistance in the laboratory. Furthermore, Gibraltar. fi we thank M. Branco, D. James Harris and R. Xavier for providing valu- At rst sight, these results support the idea of limited dis- able comments and suggestions. David James Harris conducted the Eng- persal abilities in C. penantis. Restricted dispersal may indirectly lish revision of the text. Financial support of this study was provided by enhance differentiation among populations by promoting the the Ministerio de Educacion y Ciencia (Ministry of Education and Sci- effects of local selection, inbreeding and drift (Pelc et al. 2009). ence) (Projects CGL2007-60044/BOS and CGL2011-2274) cofinanced by However, the high population connectivity found in the same FEDER funds, and by the Consejería de Innovacion, Ciencia y Empresa, region for other species with direct development (Xavier et al. Junta de Andalucía (Department of Innovation, Science and Enterprise of 2012) as well as the existence of a genetic break at Cape Ghir Andalusia) (Projects P07-RNM-02524 and P11-RNM-7041). P. Cabezas was supported by a fellowship from the Fundacion Caja Madrid. M. P. (approximately 250 km south of the southernmost site included ‘ in this study, Safi) in a species with larval dispersal (Jaziri and Cabezas was supported by a PhD grant III Plan Propio de Investigac- ion ’, from the University of Seville. Benazzou 2002), suggest that dispersal ability is probably not the only explanation for the patterns observed in this study. Probably the existence of refugial areas also plays an important References role in maintaining this kind of structure. Geographical ranges of fi marine organisms are altered by climate changes (Lima et al. Akaike H (1974) A new look at the statistical model identi cation. IEEE Trans Autom Control 19:716–723. 2007), which force organisms into refugial areas with consequent Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic events of retreat/expansion of species distributional ranges that local alignment search tool. 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