To refer to this article use this url: http://contributionstozoology.nl/vol79/nr03/a04


Contributions to Zoology, 79 (3) – 2010

Phylogeography, genetic diversity and structure of the poecilosclerid sponge Phorbas fictitius at oceanic islands

Joana R. Xavier1,2,3,4,5, Rob W.M. van Soest2, Johannes A.J. Breeuwer1, António M.F. Martins3, Steph B.J. Menken1

1.  Evolutionary Biology, Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, Kruislaan 318, 1098 SM Amsterdam, The Netherlands

2.  Netherlands Centre for Biodiversity Naturalis, section Zoological Museum of Amsterdam (ZMA), University of Amsterdam, Mauritskade 57, 1092 AD Amsterdam, The Netherlands

3.  CIBIO - Research Centre in Biodiversity and Genetic Resources, CIBIO-Azores, Department of Biology, University of the Azores, Rua Mãe de Deus, 9501-801 Ponta Delgada, Portugal (current address J.R. Xavier)

4.  CEAB - Centre for Advanced Studies of Blanes (CSIC), Camí d’accés a la Cala S. Francesc, 14, 17300 Blanes (Girona), Spain (current address J.R. Xavier)

5. joanarxavier@gmail.com

Keywords: Azores, genetic diversity, Iberia, Macaronesia, mitochondrial DNA, Porifera

Abstract


In this study we assessed the sequence variation in the I3-M11 partition of the mtDNA cytochrome c oxidase subunit I gene (COI) in ten populations of the Atlanto-Mediterranean demosponge Phorbas fictitius (Porifera: Poecilosclerida) at two spatial scales: a regional scale comparing mainland (Iberian) and insular (Macaronesian) populations, and a local (Archipelagic) scale focusing on different island populations of the Azores archipelago. A multiple approach combining diversity measures, FST estimates, phylogenetic inference and nested clade phylogeographic analysis was used to assess the genetic structure and elucidate the evolutionary history of this species. Genetic differentiation, based of FST estimates, was found among most populations at both scales revealing highly structured populations. This results of a presumably low dispersal potential and bathymetric range of the species, and the geographical isolation of the studied populations. However we found evidence of long distance dispersal events between some populations. Phylogenetic and network analyses indicate a separation of insular (Macaronesian) and mainland (Iberian) clades with only two haplotypes shared between these areas. The high genetic diversity and prevalence of ancestral haplotypes suggest the Macaronesian islands as the likely place of origin of this species with posterior expansion to mainland locations via current-mediated dispersal of larvae or sponge fragments. This study adds to the growing evidence of structured populations in the marine realm and highlights the importance of the Macaronesian islands on the evolutionary history of the Northeast Atlantic marine biota.

Introduction


Understanding the spatial patterns of genetic diversity and both the historical and contemporary factors that have shaped such genetic structure is crucial for the development of effective conservation strategies in the increasingly threatened marine realm. The Northeast Atlantic and Mediterranean region provides an interesting as well as challenging model area to study these topics for several reasons: it presents an extremely diverse and relatively well studied biota; it encompasses a wide range of subtropical, temperate, and subarctic climatic conditions; it possesses some distinguishable putative physical barriers (e.g. Strait of Gibraltar, English Channel); and it experienced a complex geological and climatological history in both the recent (e.g. Last Glacial Maximum, 30-19 kyr BP) as well as the remote past (e.g. the Messinian Salinity Crisis, 5.9-5.3 Myr BP).

Over the past decade, the phylogeography and population genetics of a great variety of marine organisms throughout the Northeast Atlantic and Mediterranean has been the focus of many investigations. These studies uncovered, among other things, the influence of physical barriers, biological traits, and past climate on the structuring of current patterns of genetic diversity and divergence among populations (see reviews in Patarnello et al., 2007; Maggs et al., 2008). However, most studies have focused on the mainland shores and only few examined populations of the Macaronesian islands (e.g. Domingues et al., 2006, 2007b, 2007c, 2008; Chevolot et al., 2006).

Sponges constitute a dominant group in hard-bottom benthic communities both in terms of biomass and species richness (Sarà and Vacelet, 1973). They are sessile in the adult phase and only disperse by means of lecitotrophic larvae with a life span of a few days to two weeks (Maldonado, 2006). Although passive dispersal by water currents may occur, most sponge larvae appear to remain in the immediate vicinity of the parental location (Mariani et al., 2005, 2006). This low dispersal potential has important consequences for the connectivity and structure of sponge populations. Surprisingly, studies addressing the population genetics and phylogeography of sponges are still scarce and limited to a handful of species worldwide (e.g. Wörheide et al., 2002; Duran et al., 2004a; Wörheide, 2006; Bentlage and Wörheide, 2007; Wörheide et al., 2008; Blanquer et al., 2009; DeBiasse et al., 2010). In the Northeast Atlantic and Mediterranean areas, and despite a remarkable diversity of 700+ shallow-water species, studies into the phylogeography of sponges are restricted to a single species, the Mediterranean Crambe crambe, that also occurs at some locations in adjacent Atlantic waters. In a first study, Duran and co-workers reported low sequence variation of the cytochrome c oxidase subunit I gene (COI) in sponges (Duran et al., 2004c). In fact, with only two mtDNA haplotypes detected in samples spanning over 3,000 km, Folmer’s COI fragment (Folmer et al., 1994) in sponges proved to be amongst the slowest evolving ones reported for marine organisms. Although some genetic structure was found among Atlantic and Mediterranean populations, this gene fragment failed to reveal the phylogeographic history of the species. Soon after, sensitivity was increased by sequencing the nuclear rDNA internal transcribed spacers (ITS-1 and ITS-2) and microsatellite genotyping for the same specimens and a recent origin of the species or, alternatively, a recent bottleneck followed by a range expansion from the Mediterranean to the Macaronesian islands by human-mediated transport was then proposed (Duran et al., 2004a, 2004b).

FIG2

Fig. 1. Geographical distribution of mtDNA haplotypes of Phorbas fictitius at (A) regional (Iberian/Macaronesian) and (B) local (Archipelagic) scales. Letters in parentheses refer to population codes (see Table 1).

In the present study, we examined the genetic structure and phylogeographical history of another poecilosclerid sponge, the Atlanto-Mediterranean Phorbas fictitius (Bowerbank, 1866), based on mtDNA sequences of an alternative partition of the COI gene (‘I3-M11’) proposed to be suitable to infer intraspecific relationships in Porifera (Erpenbeck et al., 2006; López-Legentil and Pawlik, 2009). Phorbas fictitius is an encrusting shallow-water sponge typical of the rocky subtidal. It has a wide distribution range in the Northeast Atlantic (from the West coast of Scotland to the Canaries) and Mediterranean (from Alboran to the Aegean Sea). No specific information is available about the reproductive ecology of P. fictitius, but members of the family Hymedesmiidae, to which this species belongs, are known to release brooded larvae to the surrounding water (Maldonado, 2006). The main goal of this study was to assess the extent of genetic differentiation and structure of P. fictitius populations at regional [Iberian (mainland) versus Macaronesian (island) populations] and local (populations of different islands of the Azores archipelago) scales.

Material and methods


Sampling

Specimens of P. fictitius (total n = 94) were collected by scuba-diving at ten locations separated by distances ranging from 55 to 3250 km (Fig. 1, Table 1). Specimens were preserved in 96% ethanol and deposited in the Porifera collection of the Zoological Museum of Amsterdam (ZMAPOR, now Netherlands Centre for Biodiversity Naturalis). Small fragments (3 mm3) to be used for genetic analyses were preserved in absolute ethanol and kept at -10oC until further processing.

FIG2

Table 1. Diversity measures for Phorbas fictitius populations. Population code (Pc), sample size (N), number of haplotypes (Nh), haplotype diversity (Hd), and nucleotide diversity (π) are presented. Standard deviations for Hd and π are given in parenthesis. GAL, BER and MED correspond to the Iberian populations while MAD, CAN and AZO constitute the Macaronesian populations.

DNA extraction, amplification and sequencing

Total DNA was extracted using DNeasy® Tissue kit (QIAGEN), following manufacturer instructions, and a fragment of the COI gene was sequenced. Folmer’s COI partition (Folmer et al., 1994) has an extremely slow rate of sequence evolution in sponges (Duran et al., 2004c, Wörheide, 2006) and anthozoans (Shearer et al., 2002; France and Hoover, 2002), therefore exhibiting a low resolution in the assessment of relationships at inter- and intraspecific levels in these groups. However Erpenbeck et al. (2006) showed that COI can be a suitable marker if another partition (‘I3-M11’) located downstream of Folmer’s fragment is used. In this study, we amplified and sequenced a partition of the COI gene that overlaps approximately 60bp with Folmer’s 3’ partition and includes Erpenbeck’s ‘I3-M11’. For that purpose we designed a new primer set from the alignment of three complete poriferan COI sequences available from Genbank (NC006894, NC006990, NC006991; Lavrov et al., 2005) with our own sequences. These primers, PficCOI2 f (5’ –AACATGAGGGCANTGGGAGTAACT– 3’) and PorCOI2 r (5’ –ACTGCCCCCATNGATAAAACAT– 3’), were developed for P. fictitius but also appear to successfully amplify and sequence COI from sponge species belonging to orders different than the Poecilosclerida (e.g. Hadromerida, Astrophorida).

Amplifications were carried out in 25 µl volume reaction containing 2.5 µl of 10x buffer (Sphaero Q), 4 µl dNTPs (1 mM), 1.6 µl BSA (10 mg/ml), 1.6 µl MgCl2 (25 mM), 0.3 µl (5 U/µl) of Taq polymerase (Sphaero Q), 0.8 µl of each primer (10 µM), and 1.5 µl of DNA. The amplification profile was as follows: initial denaturing step of 95oC for 3 min, 36 cycles (94oC for 30 s, 57oC for 45 s and 70oC for 90 s) and a final extension of 72oC for 10 min. Amplified products were excised from 1% TAE gels and purified with QIAquick Gel Extraction kit (QIAGEN) following the manufacturer’s instructions. The same primers were used for the sequencing reaction with the ABI-Big-Dye Ready-Reaction and purified products sequenced on both directions on an ABI 3700 automated sequencer at the Amsterdam Academic Medical Centre.

Data analyses

Multiple alignments were performed using the ClustalW tool in BioEdit (version 7.0.0, Hall 1999).

Genetic diversity and structure

Haplotype and nucleotide diversities were calculated for each population in DnaSP (version 4.0; Rozas et al., 2003). Genetic differentiation among populations was assessed from pairwise FST analyses and gene flow (M) estimates. Analysis of molecular variance (AMOVA) was performed in order to assess the hierarchical population structure at the considered spatial scales. At the regional scale, all Azorean populations were pooled and compared with the other Iberian populations. At the archipelagic scale, we performed an AMOVA exclusively for the five Azorean populations to assess the level at which island populations of P. fictitius are structured. In order to test for a model of isolation by distance we applied a Mantel test to the pairwise genetic and geographical distance matrices. All analyses were implemented in ARLEQUIN (version 3.11, Excoffier et al., 2005).

Phylogenetic and phylogeographic relationships

Phylogenetic relationships among haplotypes were assessed under Maximum Parsimony (MP) by full heuristic search and the confidence was evaluated with 5,000 replicates in PAUP* (version 4.0, Swofford, 1998). To infer the pattern of historical processes that may have shaped the current distribution range of this species, we performed nested clade phylogeographic analyses (NCPA, Templeton et al., 1995; Templeton, 1998, 2004) which has proved to be a useful technique to assess phylogeographic relationships in two other sponge species (Wörheide et al., 2002; Duran et al., 2004a). We are aware of the recent debate on the effectiveness of this inference method (see Knowles, 2008; Petit, 2008; but also Templeton, 2008, 2009) and for that reason we used NCPA as an additional and not an exclusive analytical approach to our data. In order to avoid subjective interpretations during the phylogeographic inference, the NCPA analysis was performed in ANeCA version 1.2. which is a fully automated implementation of this method (Panchal, 2007). This software uses TCS v1.21. to build a haplotype network through implementation of a statistical parsimony criterion (Clement et al., 2000), and GeoDis version 2.5. that tests for the geographical association of haplotypes through calculation of nested clade statistics and their significance (Posada et al., 2000, 2006).

[ *image not found: m7903a04:warning-no-figure-id-found ]

Results


Sequence variation and genetic diversity

A total of 94 partial COI sequences were obtained for P. fictitius with an aligned length of 557 bp. These resulted in ten haplotypes defined by nine variable sites, deposited in GenBank under Accession nos. GQ273482 – GQ273491. Distances between haplotypes ranged from 0.18% to 1.28% and the highest number of substitutions found was seven (between haplotypes I and VI).

Overall haplotype and nucleotide diversities were 0.747 and 0.0042, respectively. At the regional scale, island (Macaronesian) populations revealed higher genetic diversity than their mainland (Iberian) counterparts. A gradient of lower diversity at higher latitude was observed. The northern (Galicia) and easternmost (Mediterranean) populations revealed the lowest haplotype and nucleotide diversities. At the archipelagic scale, haplotypic diversity ranged from 0 at the westernmost islands (Flores and Faial islands) to 0.509 at the eastern island group (São Miguel island) (Table 1).

Phylogenetic and phylogeographic relationships

The geographical distribution of haplotypes at the two spatial scales is shown in Fig. 1. Of the ten detected haplotypes, two (haplotypes I and II) are confined to the northernmost Iberian populations (Galicia and Berlengas; Fig. 1a). Haplotype III is shared between Berlengas and all the Macaronesian archipelagos (Madeira, Canaries, and Azores) and haplotype IV is shared between the latter and the only Mediterranean population (Blanes). Six private haplotypes were detected, one in the Azores (X), two in Madeira (VI and VII) and the Canaries (VIII and IX), and one in the Mediterranean (V) populations. Several haplotypes are prevalent (relative frequency >0.6) in some populations: haplotype I in Galicia, IV in the Canaries and Azores, V in the Mediterranean, and haplotype VII in Madeira (Table 2). The distribution of haplotypes at the archipelagic scale was characterized by the presence of a single (and different) haplotype on the islands of Faial (IV) and Flores (III), in the westernmost part of the archipelago (Fig. 1b).

FIG2

Fig. 2. Phylogenetic relationships of Phorbas fictitius haplotypes: (A) unrooted Maximum Parsimony consensus tree. Only bootstrap support values that are >50% are shown; (B) Statistical parsimony network. The area of the polygons is prportional to the frequency of the haplotypes in the total sample; (C) Nested clade design.

Due to the low sequence variation, the analysis of phylogenetic relationships between haplotypes resulted in a moderately supported tree, with bootstrap values just above 60 (Fig. 2a). However, both the phylogenetic tree and the statistical parsimony network (Fig. 2b) showed the same topology: a clade comprising haplotypes IV, VI, VII, and X with a southern (insular) distribution and another one comprising haplotypes I and II restricted to the northern (mainland) locations. Haplotype IV, occurring in all three Macaronesian archipelagos (AZO, CAN, MAD) and in the Mediterranean population (MED) was found to be the ancestral haplotype it yield the highest outgroup probability (0.230).

The statistical parsimony analysis revealed a network with haplotypes III and IV in a central position and from which all other haplotypes derive by few mutations. The nested clade design estimated seven 1-step clades and three 2-step clades. Significant associations between haplotypes and geographical distribution were found at several levels but inferences were inconclusive except for two 2-step level clades. Restricted gene flow/dispersal but with some long-distance dispersal over intermediate areas not occupied by the species or past gene flow followed by extinction of intermediate populations was inferred between the mainland populations of Galicia and Berlengas and the Macaronesian islands (clade 2-1), whereas long distance colonization and/or past fragmentation was the process inferred between the islands and Mediterranean populations (clade 2-2) (Fig. 2c, Table 5).

FIG2

Table 2. Relative frequencies of ten COI haplotypes in each of six Phorbas fictitius populations. Population abbreviations as in Table 1.

FIG2

Table 3. Pairwise FST values (below diagonal) and gene flow estimates M (above diagonal) between Phorbas fictitius populations at regional (Iberian/Macaronesian - top) and local (Archipelagic - bottom) scales. * significant values at P<0.05, ns - not significant, inf. - infinite.

FIG2

Table 4. Analysis of molecular variance (AMOVA) for COI sequences of Phorbas fictitius at two spatial scales. At the regional scale (Iberian/Macaronesian) the Azorean populations were pooled and at the local (Archipelagic) scale only Azorean populations are considered. Va and Vb represent the associated covariance components. Significant values of FST (P<0.001) are indicated with an asterisk.

FIG2

Table 5. Nested contingency analysis of geographical association of the clades and biological inference from the NCPA. Χ2 is the observed chi-square statistics and P is the probability of a random Χ2 being greater than or equal to the observed value after 10,000 resamples.

Differentiation and structure at regional and local scales

The genetic structure of P. fictitius populations was first assessed by pairwise FST and gene flow estimates (Table 3). Significant high levels of genetic differentiation were found between most population pairs at the regional spatial scale. The northernmost population (GAL) showed the highest differentiation to all other populations (FST >0.7) with the exception of Berlengas (500 km to the South), to which it showed no significant differentiation. The lowest differentiation (FST <0.4) was found among the Macaronesian archipelagos (AZO/MAD/CAN) with the Azorean populations showing no significant differentiation to the Canarian ones.

At the archipelagic scale, two contrasting results stand out: high FST values between populations that are only tens of kilometres apart (FST >0.5 between SMA/FOR and SMG) versus non-significant differentiation between populations that are hundred’s of kilometres apart (for instance, FST = 0.207 between SMG and FLW that are over 500 km apart). The low sequence variation and limited sampling may cause an overestimation of the FST values and therefore these have to be regarded cautiously. However, population differentiation is evident from the geographical distribution and frequency of the haplotypes. Structure at both spatial scales was further confirmed by the AMOVA results (highly significant FST values; Table 4). At the Iberian scale, variation was similar within and among populations (FST ≈0.5) while at the archipelagic scale 71% of the total variation was found among islands.

The Mantel test, performed at the regional scale, revealed a non-significant trend (r = 0.287, P = 0.209) of increasing genetic differentiation with increasing geographical distance between populations. The same test was again not significant at the archipelagic scale (r = 0.266, P = 0.150) and therefore the observed genetic patterns could not be explained by a model of isolation by distance.

Discussion


Sequence variation and the use of an alternative partition of COI

Our results confirm the previously reported low sequence variation for mtDNA in sponges. Nonetheless, the overall nucleotide diversity (π = 0.0042) found in the ‘I3-M11’ partition in our study was higher but of the same magnitude as the value found for the giant barrel sponge in the Caribbean (Xestospongia muta, π = 0.0039, López-Legentil and Pawlik, 2009) using this same partition, and much higher than the values found in Folmer’s COI partition in several species at similar but also larger spatial scales (Crambe crambe, π = 0.0006, Duran et al., 2004c; Astrosclera willeyana, π = 0.00049, Wörheide, 2006; Xestospongia muta, π = 0.00058, López-Legentil and Pawlik, 2009).

The intraspecific variation (1.28%) found in our study is similar to the one found in Xestospongia muta I3-M11 (0.92%, López-Legentil and Pawlik, 2009) and much higher than the values found in Folmer’s COI partition for both X. muta and C. crambe (0.18%, López-Legentil and Pawlik, 2009; and 0.19%, Duran et al., 2004c, respectively). Our findings therefore support that this alternative partition of the COI gene, located downstream of Folmer’s partition, is indeed more suitable to infer interspecific relationships in sponges as initially suggested by Erpenbeck et al. (2006) and even for intraspecific studies but for species with somewhat deeper phylogeographic histories. Furthermore, the use of taxon-specific primers has obvious methodological advantages over the use of ‘universal’ primers such as those of Folmer (Folmer et al., 1994), particularly in groups like sponges that are known to host diverse microbial communities (Hentschel et al., 2003). The primers developed by us amplify a large range of sponge species belonging to different orders and therefore we highly recommend its use for lower level phylogenetic studies in this taxonomic group.

Structure of P. fictitius at regional and local scales

Despite the low sequence variation, we found P. fictitius to have highly structured populations at both regional and local spatial scales, as evidenced by the pairwise FST values and the AMOVA results. This structure is consistent with the low dispersal potential and bathymetric range of the species. Previous studies have shown sponge larvae to be philopatric and to recruit at short distance from the parental locations (Mariani et al., 2005, 2006). Furthermore P. fictitius is a shallow-water species inhabiting rocky habitats down to 50 m depth and therefore oceanic depths may constitute a strong barrier to gene flow and range expansion in this species. Similar evidence of genetically structured populations has been found for the demosponge C. crambe at comparable spatial scales in the same area (Duran et al., 2004a, 2004b). The same pattern was also observed in the giant barrel Xestospongia muta in the Caribbean (López-Legentil and Pawlik, 2009) and the common reef sponge Callyspongia vaginalis along the Florida reef tract (DeBiasse et al., 2010). Moreover, a strong fine-scale (from cm to m) genetic structure was also observed in both Crambe crambe (Calderón et al., 2007) and Scopalina lophyropoda (Blanquer et al., 2009) in the Mediterranean. Together these studies suggest that structured populations are to be expected in most sponge species at various spatial scales as a result of a presumed limited dispersal potential of their lecitotrophic larvae.

However, we observed a non-significant differentiation between Berlengas and Galicia as well as between the Azores and the Canaries, separated by 500 km and 1500 km, respectively. These observations suggest that although P. ficittius populations are highly structured as a result of restricted dispersal there may be occasional long-distance dispersal events between some populations. Since very few studies have directly evaluated larval dispersal in sponges this possibility cannot be discounted. Furthermore, Maldonado and Uriz (1999) have shown that small fragments of reproductive sponges, containing embryos, broken by wave or predatory action can be transported by currents and recruit to a new area. Since P. fictitius is a eurytopic species, i.e. with a great plasticity in adapting to a wide variety of environmental conditions (Carballo et al., 1996) it is likely that fragments could thrive during such dispersal events. This long distance dispersal, also inferred in the nested clade analysis, would explain why a pattern of isolation by distance was not found at neither scales. Similar evidence of occasional long dispersal events, and lack of isolation by distance, was found in populations of Callyspongia vaginalis along the Florida reef tract (DeBiasse et al., 2010).

At the archipelagic scale, we found a very patchy distribution of mtDNA haplotypes. However, the differentiation that we found between most population pairs even at spatial scales of the order of tens of km suggests structured and therefore non-panmictic archipelagic populations that would otherwise exhibit more homogenous haplotype distribution and frequency. The absence of genetic diversity in the populations from Flores and Faial islands may indicate a recent expansion of the species, via a founder event, to the westernmost part of the archipelago or a population bottleneck. However, sampling of these islands was limited (n = 6 and n = 7) and therefore variation could have been missed. A more intensive sampling in these and the remaining islands will be necessary to confirm whether habitat discontinuity by the deep-sea promotes genetic subdivision in shallow-water species of island ecosystems as proposed by some authors. Such patterns of island subdivision have been found for the antherinid fish Craterocephalus capreoli (Johnson et al., 1994) and the intertidal snail Austrocochlea constricta in the Houtman Abrolhos Islands (Johnson and Black, 2006). Contrastingly, no genetic differentiation was found among island populations of the blackbelly rosefish Helicolenus dactylopterus, in the Azores, given the continuity of its deep-sea habitat (Aboim et al., 2005). These examples emphasize the complex interplay between intrinsic biological and ecological traits (e.g. dispersal potential, geographic and bathymetric range, substrate preference) and extrinsic present and past environmental factors (e.g. habitat continuity, geographical distance, bathymetry, prevailing surface circulation) on the structuring of the populations at diverse spatial scales.

Phylogeography of P. fictitius

Although only moderately supported, the phylogenetic reconstruction of haplotypes and the parsimony network reveals the existence of insular (Macaronesian) and mainland (Iberian) clades with only two haplotypes (III and IV) shared between these locations. The level of differentiation that we found in our study reflects the high degree of isolation among island and mainland populations of P. fictitius. A comparable isolation between the Macaronesian islands and the continental shores has been previously reported for the perciform triplefin Tripterygion delaisi (Domingues et al., 2007a) and for several limpet species of the genus Patella (Sá-Pinto et al., 2008).

Some important attributes of haplotype networks, derived from coalescent theory, have shown that haplotypes with an interior position in the network are older than haplotypes on the tips (network age polarity) and that older haplotypes are more widespread than younger haplotypes under a restricted gene flow model (haplotypes geographical range and frequency) (Castelloe and Templeton, 1994; Templeton et al., 1995; Templeton, 1998). Haplotype IV having a central position in the network and being the most geographically spread was found to be the oldest. Haplotype III although having a lower outgroup weight (0.110) is also in a central position and is equally widely distributed. Given the prevalence of these ancestral haplotypes and the highest genetic diversity observed in all island populations it seems plausible to assume these archipelagos as the putative origin of the species with posterior expansion via current mediated dispersal of larvae or sponge fragments to mainland locations (haplotype III to the Portuguese mainland and haplotype IV to the Mediterranean) followed by haplotype diversification. The island-to-mainland direction of dispersal is in our opinion more likely given the eastward flowing currents characteristic of this region such as the Azores and the North Atlantic currents (Reverdin et al., 2003). Our findings therefore contrast with those of Duran and colleagues that inferred a recent human-mediated introduction of C. crambe in the Macaronesian islands from the Mediterranean (Duran et al., 2004a).

NCPA inferences regarding the population history of P. fictitius were only possible for two clades (clade 2-1 and 2-2, Fig. 2c). In the case of clade 2-1 restricted gene-flow but with some long distance dispersal is more likely to occur between the mainland (GAL, BER) and between these and island populations since these are naturally isolated and no intermediate populations exist. This is further corroborated by the non-significant FST values found between Galicia and Berlengas. The inference made for clade 2-2, that comprises island haplotypes and the only Mediterranean haplotype, confirms the occasional long distance dispersal events observed from the non-significant FST values between Madeira and the Canaries. It therefore seems that current gene flow patterns more than historical events are the main factors shaping the genetic structure of Phorbas ficittius populations (but see next section). Further studies in other species are required if we are to better understand the phylogeographic patterns of the Northeast Atlantic and Mediterranean sponge fauna.

The Macaronesian refugium

The Pleistocene glaciations, and in particular the Last Glacial Maximum, are known to have shaped the present-day distribution and genetic structure of both terrestrial and aquatic biota in the Northeast Atlantic and Mediterranean areas. Current models of glacial refugia use genetic diversity estimates, the spatial distribution and relative ages of haplotypes to identify refugial and expansion areas. Refugia are usually characterized by possessing the highest genetic diversity (except in cases of contact zones) and by a mixture of ancestral and private haplotypes, while expansion areas are usually genetically depauperate and composed of a subset of the refugial gene pool (Hewitt, 2000; Maggs et al., 2008).

Based on these premises the Macaronesian islands have been proposed as an offshore refugium for several marine organisms such as the pomacentrid Chromis limbata (Domingues et al., 2006), the white seabream Diplodus sargus (Domingues et al., 2007c), the blennids Coryphoblennius galerita (Domingues et al., 2007b) and Parablennius parvicornis (Domingues et al., 2008), the thornback ray Raja clavata (Chevolot et al., 2006) as well as for several species of the Patella genus (Sá-Pinto et al., 2008). This is the result of largely stable climatic conditions experienced by these archipelagos during the Pleistocenic glaciations (see Crowley, 1981; Pflaumann et al., 2003, Hayes et al., 2005).

From the geographical distribution of mtDNA haplotypes of P. fictitius two observations stand out: (a) highest genetic diversity at the Macaronesian archipelagos and a latitudinal gradient in diversity (highest diversity at southern locations) and (b) high frequency of ancestral (haplotype IV) and private haplotypes at all three Macaronesian archipelagos. Combined, these observations suggest the Macaronesian islands may have served as putative offshore refugia for P. fictitius populations. However, data from additional Mediterranean and northern European populations would be required to further corroborate this hypothesis.

Acknowledgements


The authors wish to thank the following colleagues and respective institutions for their support during the sampling work: the Departments of Biology (A. Costa) and Oceanography and Fisheries (F. Tempera, F. Cardigos, R. Santos), University of the Azores; Marine Biological Station of Funchal (M. Biscoito), Marine Biological Station of Graña (J. Cristobo, P. Rios), and Centre de Estudis Avançats de Blanes (A. Blanquer, M. Uriz); in Tenerife (T. Cruz, L. Moro, J. Bacallado). We thank Rosa Pestana and Andreia Cunha for providing some additional samples from Madeira and São Miguel Islands. We further thank Wil van Ginkel, Betsie Voetdijk, and Peter Kuperus for their valuable help in the molecular lab. We also warmly acknowledge M. Veith for his guidance with the use of some software packages, Paola Rachello-Dolmen for her help in the construction of Fig. 1, and Julie Reveillaud and Nuno Curado for useful comments on an earlier version of the manuscript. This study is part of the PhD-project of J. Xavier, funded by Fundação para a Ciência e Tecnologia (FCT-Portugal, grant no. SFRH/BD/16024/2004).

Received: 17 June 2009

Revised and accepted: 31 August 2010

Published online: 1 October 2010

Editor: J.W. Arntzen

References


Aboim MA, Menezes GM, Schlitt T, Rogers AD. 2005. Genetic structure and history of populations of the deep-sea fish Helicolenus dactylopterus (Delaroche, 1809) inferred from mtDNA sequences analysis. Molecular Ecology 14: 1343-1354.

Bentlage B, Wörheide G. 2007. Low genetic structuring among Pericharax heteroraphis (Porifera: Calcarea) populations from the Great Barrier Reef (Australia), revealed by analysis of nrDNA and nuclear intron sequences. Coral Reefs 26: 807-816.

Blanquer A, Uriz MJ, Caujapé-Castells J. 2009. Small-scale spatial genetic structure in Scopalina lophyropoda, an encrusting sponge with philopatric larval dispersal and frequent fission and fusion events. Marine Ecology Progress Series 380: 95-102.

Bowerbank JS. 1866. A Monograph of the British Spongiadae, volume 2. London: Ray Society.

Calderón I, Ortega N, Duran S, Becerro M, Pascual M, Turon X. 2007. Finding the relevant scale: clonality and genetic structure in a marine invertebrate (Crambe crambe, Porifera). Molecular Ecology 16: 1799-1810.

Carballo J, Naranjo S, Garcia-Gomez J. 1996. Use of marine sponges as stress indicators in marine ecosystems at Algeciras Bay (southern Iberian Peninsula). Marine Ecology Progress Series 135: 109-122.

Castelloe J, Templeton AR. 1994. Root probabilities for intraspecific gene trees under neutral coalescent theory. Molecular Phylogenetics and Evolution 3: 102-113.

Chevolot M, Hoarau G, Rijnsdorp A, Stam W, Olsen J. 2006. Phylogeography and population structure of thornback rays (Raja clavata L., Rajidae). Molecular Ecology 15: 3693-3705.

Clement M, Posada D, Crandall K. 2000. TCS: a computer program to estimate gene genealogies. Molecular Ecology 9: 1657-1660.

Crowley T. 1981. Temperature and Circulation changes in the Eastern North Atlantic during the last 150,000 years: evidence from the planktonic foraminiferal record. Marine Micropaleontology 6: 97-129.

DeBiasse MB, Richards VP, Shivji MS. 2010. Genetic assessment of connectivity in the common reef sponge, Callyspongia vaginalis (Demospongiae: Haplosclerida) reveals high population structure along the Florida reef tract. Coral Reefs 29: 47-55.

Domingues VS, Santos RS, Brito A, Almada VC. 2006. Historical population dynamics and demography of the eastern Atlantic pomacentrid Chromis limbata (Valenciennes, 1833). Molecular Phylogenetics and Evolution 40: 139-147.

Domingues VS, Almada VC, Santos RS, Brito A, Bernardi G. 2007a. Phylogeography and evolution of the triplefin Tripterygion delaisi (Pisces, Blennioidei). Marine Biology 150: 509-519.

Domingues VS, Faria C, Stefanni S, Santos RS, Brito A, Almada VC. 2007b. Genetic divergence in the Atlantic-Mediterranean Montagu’s blenny, Coryphoblennius galerita (Linnaeus 1758) revealed by molecular and morphological characters. Molecular Ecology 16: 3592-3605.

Domingues VS, Santos RS, Brito A, Alexandrou M, Almada VC. 2007c. Mitochondrial and nuclear markers reveal isolation by distance and effects of Pleistocene glaciations in the northeastern Atlantic and Mediterranean populations of the white seabream (Diplodus sargus, L.). Journal of Experimental Marine Biology and Ecology 346: 102-113.

Domingues VS, Stefanni S, Brito A, Santos RS, Almada VC. 2008. Phylogeography and demography of the Blenniid Parablennius parvicornis and its sister species P. sanguinolentus from the northeastern Atlantic Ocean and the western Mediterranean Sea. Molecular Phylogenetics and Evolution 46: 397-402.

Duran S, Giribet G, Turon X. 2004a. Phylogeographical history of the sponge Crambe crambe (Porifera, Poecilosclerida): range expansion and recent invasion of the Macaronesian islands from the Mediterranean Sea. Molecular Ecology 13: 109-122.

Duran S, Pascual M, Estoup A, Turon X. 2004b. Strong population structure in the marine sponge Crambe crambe (Poecilosclerida) as revealed by microsatellite markers. Molecular Ecology 13: 511-522.

Duran S, Pascual M, Turon X. 2004c. Low levels of genetic variation in mtDNA sequences over the western Mediterranean and Atlantic range of the sponge Crambe crambe (Poecilosclerida). Marine Biology 144: 31-35.

Erpenbeck D, Hooper JNA, Wörheide G. 2006. CO1 phylogenies in diploblasts and the ‘Barcoding of Life’ – are we sequencing a suboptimal partition? Molecular Ecology Notes 6: 550-553.

Excoffier L, Laval G, Schneider S. 2005. ARLEQUIN ver. 3.0: An integrated software package for population genetics data analysis. Evolutionary Bioinformatics Online 1: 47-50.

Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R. 1994. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology 3: 294-299.

France S, Hoover L. 2002. DNA sequences of the mitochondrial COI gene have low levels of divergence among deep-sea octocorals (Cnidaria: Anthozoa). Hydrobiologia 471: 149-155.

Hall T, 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series 41, 95-98.

Hayes A, Kucera M, Kallel N, Sbaffi L, Rohling EJ. 2005. Glacial Mediterranean Sea surface temperatures based on planktonic foraminiferal assemblages. Quaternary Science Reviews 24: 999-1016.

Hentschel U, Fieseler L, Wehrl M, Gernert C, Steinert M, Hacker J, Horn M. 2003. Microbial diversity of marine sponges. Progress in Molecular and Subcellular Biology 37: 59-88.

Hewitt GM. 2000. The genetic legacy of the Quaternary Ice Ages. Nature 405: 907-913.

Johnson MS, Watts R, Black R. 1994. High levels of genetic subdivision in peripherally isolated populations of the antherinid fish Craterocephalus capreoli in the Houtman Abrolhos Islands, Western Australia. Marine Biology 119: 179-184.

Johnson MS, Black R. 2006. Islands increase genetic subdivision and disrupt patterns of connectivity of intertidal snails in a complex archipelago. Evolution 60: 2498-2506.

Knowles LL. 2008. Why does a method that fails continue to be used? Evolution 62: 2713-2717.

Lavrov DV, Forget L, Kelly M, Lang BF. 2005. Mitochondrial genomes of two demosponges provide insights into and early stage of animal evolution. Molecular Biology and Evolution 22: 1231-1239.

López-Legentil S, Pawlik JR. 2009. Genetic structure of the Caribbean giant barrel sponge Xestospongia muta using the I3-M11 partition of COI. Coral Reefs 28: 157-165.

Maggs CA, Castilho R, Foltz D, Henzler C, Jolly MT, Kelly J, Olsen J, Perez KE, Stam W, Väinölä, Viard F, Wares J. 2008. Evaluating signatures of glacial refugia for North Atlantic benthic marine taxa. Ecology 89: 108-122.

Maldonado M. 2006. The ecology of sponge larvae. Canadian Journal of Zoology 84: 175-194.

Maldonado M, Uriz MJ. 1999. Sexual propagation by sponge fragments. Nature 398: 476.

Mariani S, Uriz M, Turon X. 2005. The dynamics of sponge larvae assemblages from northwestern Mediterranean nearshore bottoms. Journal of Plankton Research 27: 249-262.

Mariani S, Uriz M, Turon X, Alcoverro T. 2006. Dispersal strategies in sponge larvae: integrating the life history of larvae and the hydrologic component. Oecologia 149: 174-184.

Panchal M. 2007. The automation of Nested Clade Phylogeographic Analysis. Bioinformatics 23: 509-510.

Patarnello T, Volckaert FAMJ, Castilho R. 2007. Pillars of Hercules: is the Atlantic-Mediterranean transition a phylogeographical break? Molecular Ecology 16: 4426-4444.

Petit RJ. 2008. The coup de grâce for the nested clade phylogeographic analysis? Molecular Ecology 17: 516-518.

Pflaumann U, Sarnthein M, Chapman M, d’Abreu L, Funnell B, Huels M, Kiefer T, Maslin M, Schulz H, Swallow J, Kreveld S, Vautravers M, Vogelsang E, Weinelt M. 2003. Glacial North Atlantic: sea-surface conditions reconstructed by GLAMAP 2000. Paleoceanography 18: 1065-1093.

Posada D, Crandall KA, Templeton AR. 2000. GEODIS: a program for the cladistic nested clade analysis of the geographical distribution of genetic haplotypes. Molecular Ecology 9: 487-488.

Posada D, Crandall KA, Templeton AR. 2006. Nested clade analysis statistics. Molecular Ecology Notes 6: 590-593.

Reverdin G, Niiler PP, Valdimarsson H. 2003. North Atlantic Ocean surface currents. Journal of Geophysical Research 108: 3002.

Rozas J, Sanchez-Delbarrio J, Messeguer X, Rozas R. 2003. DnaSP, DNA polymorphism analyses by the coalescent and other methods. Bioinformatics 19: 2496-2497.

Sá-Pinto A, Branco M, Sayanda D, Alexandrino P. 2008. Patterns of colonization, evolution and gene flow in species of the genus Patella in the Macaronesian Islands. Molecular Ecology 17: 519-532.

Sarà M, Vacelet J. 1973. Écologie des Démosponges. Pp 462-575 in: Grassé P, Traité de Zoologie, Spongiaires. Paris: Masson et Cie.

Shearer T, Van Oppen MJH, Romano S, Wörheide G. 2002. Slow mitochondrial DNA sequence evolution in the Anthozoa (Cnidaria). Molecular Ecology 11: 2475-2487.

Swofford DL. 1998. PAUP: Phylogenetic Analysis Using Parsimony and Other Methods. Sunderland (MA): Sinauer Associates.

Templeton AR. 1998. Nested clade analyses of phylogeographic data: testing hypotheses about gene flow and population history. Molecular Ecology 7: 381-397.

Templeton AR. 2004. Statistical phylogeography: methods of evaluating and minimizing inference errors. Molecular Ecology 13: 789-809.

Templeton AR. 2008. Nested clade analysis: an extensively validated method for strong phylogeographic inference. Molecular Ecology 17: 1877-1880.

Templeton AR. 2009. Statistical hypothesis testing in intraspecific phyloeography: nested clade phylogeographical analysis vs. approximate Bayesian computation. Molecular Ecology 18: 319-331.

Templeton AR, Routman E, Phillips CA. 1995. Separating population structure from population history: a cladistic analysis of the geographical distribution of mitochondrial DNA haplotypes in the tiger salamander, Ambystoma tigrinum. Genetics 140: 767-782.

Wörheide G, Hooper JNA, Degnan B. 2002. Phylogeography of western Pacific Leucetta ‘chagosensis’ (Porifera: Calcarea) from ribosomal DNA sequences: implications for population history and conservation of the Great Barrier Reef World Heritage Area (Australia). Molecular Ecology 11: 1753-1768.

Wörheide G. 2006. Low variation in partial cytochrome oxidase subunit I (COI) mitochondrial sequences in the coralline demosponge Astrosclera willeyana across the Indo-Pacific. Marine Biology 148: 907-912.

Wörheide G, Epp L, Macis L. 2008. Deep genetic divergence among Indo-Pacific populations of the coral reef sponge Leucetta chagosensis (Leucettidae): founder effects, vicariance or both? BMC Evolutionary Biology 8: 24.