European Journal of Phycology ISSN: 0967-0262 (Print) 1469-4433 (Online) Journal homepage: https://www.tandfonline.com/loi/tejp20 Taxonomic reassessment of Polysiphonia foetidissima (Rhodomelaceae, Rhodophyta) and similar species, including P. schneideri, a newly introduced species in Europe Pilar Díaz-Tapia, Myung Sook Kim, Antonio Secilla, Ignacio Bárbara & Javier Cremades To cite this article: Pilar Díaz-Tapia, Myung Sook Kim, Antonio Secilla, Ignacio Bárbara & Javier Cremades (2013) Taxonomic reassessment of Polysiphonia�foetidissima (Rhodomelaceae, Rhodophyta) and similar species, including P.�schneideri, a newly introduced species in Europe, European Journal of Phycology, 48:4, 345-362, DOI: 10.1080/09670262.2013.842655 To link to this article: https://doi.org/10.1080/09670262.2013.842655 View supplementary material Published online: 10 Oct 2013. Submit your article to this journal Article views: 615 View related articles Citing articles: 11 View citing articles Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=tejp20 Eur. J. Phycol. (2013), 48(4): 345–362 Taxonomic reassessment of Polysiphonia foetidissima (Rhodomelaceae, Rhodophyta) and similar species, including P. schneideri, a newly introduced species in Europe PILAR DÍAZ-TAPIA1, MYUNG SOOK KIM2, ANTONIO SECILLA3, IGNACIO BÁRBARA1 AND JAVIER CREMADES1 1 Coastal Biology Research Group, Facultade de Ciencias, Universidad de A Coruña, Campus da Zapateira s/n, A Coruña 15071, Spain 2 Department of Biology and Research Institute for Basic Sciences, Jeju National University, 66 Jejudaehankno, Jeju-si, Jeju-do, 690-756, Korea 3 Departamento de Biología Vegetal y Ecología, Facultad de Ciencia y Tecnología, Universidad de Basque Country, Apdo. 644, Bilbao 48080, Spain (Received 11 October 2012; revised 8 February 2013; accepted 30 April 2013) Morphological and molecular studies were carried out on two Polysiphonia with 6–9 pericentral cells from the Atlantic Iberian Peninsula. A detailed description is provided for P. foetidissima, a poorly known species originally described from the UK that is widespread and abundant in the Iberian Peninsula. Polysiphonia schneideri, originally described from Atlantic U.S.A. and Bermuda, is reported for the first time in Europe (Southern Spain). It was collected attached to man-made structures such as floating docks and artificial substrata for aquaculture and is believed to be a newly introduced species in Europe. In addition, the taxonomy of seven morphologically similar Polysiphonia was reassessed. A comparative study of type materials showed that the Mediterranean P. stuposa is morphologically different from its alleged synonym P. foetidissima. Instead, molecular and morphological evidence showed that P. foetidissima is a synonym of the widely reported (Atlantic and Pacific) P. tepida. Polysiphonia foetidissima was also shown to differ from P. brodiei, P. exilis, P. isogona and P. schneideri. Key words: alien species, biogeography, distribution, molecular phylogeny, morphology, Polysiphonia, Polysiphonia foetidissima, Polysiphonia schneideri, rbcL, red algae, taxonomy. Introduction Polysiphonia is one of the largest genera of Rhodophyta (Womersley, 1979; Mamoozadeh & Freshwater, 2012). Almost 1000 species names have been assigned to this genus at one time or another and, at present, it is thought to contain approximately 200 recognized species (Guiry & Guiry, 2012). This large number of species, along with inadequate early descriptions and a paucity of diagnostic features in dried herbarium specimens, make the verification of species from different areas a challenge (Womersley, 1979). The problem is further aggravated by the frequency and variety of species, often misidentified, that are mentioned in most ecological accounts and checklists (Womersley, 1979) and unsupported by herbarium material. On the other hand, new species of Polysiphonia continue to be described (Maggs & Hommersand, 1993; Kim & Lee, 1999; Stuercke & Freshwater, 2010; Bárbara et al. 2013). Many of these new species had previously remained unnoticed Correspondence to: Pilar Díaz-Tapia. E-mail: pdiaz@udc.es. owing to their small size, their confinement to restricted habitats, and the challenges of the morphological delimitation of pseudo-cryptic species. In some cases, Polysiphonia species have remained undescribed due to the absence of consistent diagnostic features, and it is only recently that molecular tools have provided objective data to separate them (e.g. Stuercke & Freshwater, 2010). Likewise, some introduced species of Polysiphonia closely resemble native species and require molecular analyses to identify them reliably (McIvor et al., 2001; Geoffroy et al., 2012). In this regard, numerous introductions of macroalgae to the Atlantic European coasts have been reported over the past four decades (Farnham, 1980; Maggs & Stegenga, 1999; Hewitt et al., 2007; Bárbara et al., 2008; Couceiro et al., 2011; Mineur et al., 2012). Moreover, the actual number of invaders and their impacts may have been seriously underestimated (McIvor et al., 2001). The only species of Polysiphonia sensu lato previously reported as introduced to the Atlantic Europe are Neosiphonia harveyi and P. morrowii from Atlantic North America and ISSN 0967-0262 (print)/ISSN 1469-4433 (online)/13/040345-362 © 2013 British Phycological Society http://dx.doi.org/10.1080/09670262.2013.842655 Published online 10 Oct 2013 P. Díaz-Tapia et al. Japan, respectively (Maggs & Stegenga, 1999; McIvor et al., 2001). Nonetheless, the challenges of species discrimination noted above suggest that other cases of cryptic introductions may have been overlooked within this difficult group. Two small species of Polysiphonia with 6–9 pericentral cells were found during our recent general algal collections along the Atlantic Iberian Peninsula. Their identification was problematic and we therefore undertook an integrative morphological and molecular taxonomic reassessment of P. foetidissima and P. schneideri, for which detailed redescription proved necessary, and P. isogona, P. exilis, P. kappannae, P. nizamuddinii, P. stuposa, P. tepida, and certain small and creeping forms of P. brodiei. Materials and methods Polysiphonia specimens were collected along the Atlantic Iberian Peninsula and southern France during 2002–2011 (Table S1). Polysiphonia foetidissima was collected at 28 sites along the Atlantic coasts of the Iberian Peninsula, on sand-covered rocks from the intertidal to the subtidal. The material subsequently identified as P. schneideri was collected in 2011 at two sites from the province of Cádiz, southern Iberian Peninsula. Material collected for morphological studies was preserved in 4% formalin in seawater. Microscope slides were mounted in a mixture of 20% Karo® Syrup (ACH Foods, Memphis, Tennessee, U.S.A.) and 80% distilled water. Line drawings were made using a camera lucida and photomicrographs were taken with a Olympus C-5060 digital camera mounted on a Olympus BX50 microscope (Olympus, Tokyo, Japan). Representative specimens were deposited at the herbarium of the University of Santiago de Compostela (SANT) and the herbarium of the University of the Basque Country (BIO). The type materials of Polysiphonia foetidissima, P. tepida, P. stuposa and P. exilis were studied at PC or borrowed from MICH, MEL and L, and TCD, respectively (herbarium abbreviations follow the online Index Herbariorum at http://sweetgum.nybg.org/ih/). Material collected for molecular analysis was dried in silica gel and total genomic DNA was extracted using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. The extracted DNA was stored at –20°C and used to amplify the rbcL region in two overlapping fragments with the forward primer rbcLF145 combined with the reverse primer rbcLR898, and the forward primer rbcLF762 combined with the reverse primer rbcLR1442 (Kim et al., 2010). PCR amplification was performed in a total volume of 25 µl containing 0.5 U of TaKaRa Ex Taq DNA polymerase (Takara Shuzo, Shiga, Japan), 2.5 mM of each dNTP, 2.5 µl of the 10× Ex Taq Buffer (Mg2+ free), 2 mM MgCl2, 10 pmol of each primer, and 1–10 ng of template DNA. PCR was carried out with an initial denaturation at 96°C for 4 min, followed by 35 cycles of denaturation at 94°C for 1 min, annealing at 50°C for 1 min, and extension at 72°C for 2 min, with a final extension at 72°C for 7 min. PCR products were purified using the AccuPrep PCR Purification Kit (Bioneer, Daejeon, Korea) according to the manufacturer’s instructions. Electropherogram outputs from each sample were edited with the program Chromas 1.45 (Technelysium, Queensland, 346 Australia) and rbcL sequences were aligned using BioEdit 7.1.3 (Tom Hall, Ibis Biosciences, Carlsbad, USA). Sequences were produced for 17 specimens of Polysiphonia and aligned with 30 selected sequences for other Polysiphonia species obtained from GenBank, plus two genera (Lophosiphonia and Herposiphonia) used as the outgroup. The resulting alignment was 1241 bp long and contained 462 variable (37%) and 380 (30%) phylogenetically informative sites. The best-fit nucleotide substitution model for our sequence data was determined with the likelihood ratio test implemented in ModelTest 3.7 (Posada & Crandall, 1998) and found to be a general time reversible (GTR) model with gamma correction for among-site variation (Γ) and proportion of invariable sites (I). To confirm the taxonomic position of the two species of Polysiphonia, maximum likelihood (ML) analyses were performed with PAUP 4.0 (Swofford, 2002). ML trees were estimated using a heuristic search with 100 random addition sequence replicates, and TBR branch swapping. ML bootstrap analyses were performed with 1000 replicates. Results Phylogenetic analysis of rbcL Twelve specimens of Polysiphonia foetidissima from the Atlantic Iberian Peninsula and southern France and one of ‘Neosiphonia’ tepida from the U.S.A. were nearly identical with minor differences of up to 0.4% divergence. One specimen of P. schneideri from the Atlantic Iberian Peninsula was grouped together with specimens from Panama, U.S.A. and Bermuda, with 0.1–0.8% intraspecific divergence. Three P. brodiei from Spain and one specimen from Ireland showed 0–0.3% intraspecific divergence. In the phylogenetic tree (Fig. 1), all specimens of Polysiphonia foetidissima, together with ‘Neosiphonia’ tepida, formed a monophyletic clade with strong support (99% for ML). The ML analysis of rbcL data thus suggested that P. foetidissima was conspecific with the N. tepida from North Carolina and both were clearly separated from other species. Polysiphonia foetidissima was sister to P. isogona with 1.7–1.8% interspecific divergence. Monophyly of three other species from Spain was supported by strong bootstrap values: 100% for each of P. schneideri, P. elongata and P. brodiei. Polysiphonia schneideri formed the sister group (80% bootstrap support) to a clade comprising P. forfex, P. strictissima and N. harveyi. Sequences of P. elongata and P. elongella were 5.8% divergent and their sister relationship was supported by a moderate bootstrap value (82%). A sister relationship of P. brodiei and P. fibrillosa from Ireland had 100% bootstrap support and their sequences were 3.2–3.4% divergent. Morphological observations Polysiphonia foetidissima Cocks ex Bornet 1892, p. 314 SYNONYMS: Polysiphonia tepida Hollenberg (1958); Neosiphonia tepida (Hollenberg) S.M. Guimarães & Polysiphonia foetidissima and P. schneideri in Europe 347 Fig. 1. Maximum likelihood tree for Polysiphonia and their relatives from plastid-encoded rbcL sequence data. The bootstrap values shown above the branches are from 1000 bootstrap resamplings. M.T. Fujii in Guimarães et al. (2004; but see our discussion of the nature of the material studied by Guimarães et al.); ?Polysiphonia kappannae Sreenivasa Rao (1967); ?Polysiphonia nizamuddinii Farooqui & Begum (1978). LECTOTYPE: PC 0146017 (Figs 2–6), Herb. Cocks ‘Algarum Fasciculi’ no. 29 (Maggs & Hommersand, 1993). LECTOTYPE LOCALITY: Mount Edgcumbe, Plymouth, Cornwall, UK. Description of type material In the type material, the thalli formed dense tufts 7 cm high, consisting of interwoven prostrate and erect axes (Fig. 2); they were brownish-red. The axes were ecorticate, with seven or eight pericentral cells (Fig. 3). P. Díaz-Tapia et al. 348 Figs 2–6. Polysiphonia foetidissima: type material. 2. Lectotype (PC 0146017). 3. Cross section of an axis with 8 pericentral cells. 4. Rhizoids cut off from pericentral cells (arrow). 5. Apex showing a branch formed in the axil of a trichoblast (arrow) and trichoblasts or scar cells several segments apart (arrowheads). 6. Axes with a scar cell of a trichoblast (arrowhead). Scale bars = 1.5 cm (Fig. 2), 25 µm (Figs 3–5) and 90 µm (Fig. 6). The prostrate axes were 80–110 µm in diameter and segments were 2.2–3.8 diameters long, with rhizoids cut off from the pericentral cells (Fig. 4). The erect axes were 70–120 µm in diameter, with segments 1.7–3.3 diameters long. Branches were formed in the axils of trichoblasts at the apices of erect axes (Fig. 5), mostly at intervals of six to eight segments; the branches alternate. Trichoblasts were abundant and borne three or four segments apart in a 1/4 spiral divergence, deciduous, and left conspicuous scar cells (Fig. 6). Tetrasporangia were in spiral series in the upper parts of erect axes, one per segment. Description of material from the Iberian Peninsula Vegetative morphology: The thalli formed dense turfs up to 2 cm high, covering substratum surfaces of up to 30 cm2, and developing a large horizontal system of interwoven decumbent axes growing from erect apices (Fig. 10). The erect axes quickly became creeping by developing rhizoids from the basal segments (Fig. 7) and were alternately branched up to five orders, bearing short branches (Figs 7–8, 11). The thalli were brownish-red and erect axes had a soft and flaccid texture. The axes were ecorticate, with (six or) seven or eight (or nine) pericentral cells (Figs 13–17). Creeping axes were (60–) 90–150 (–180) µm in diameter, with segments (0.4–) 0.6–1.1 (–1.6) diameters long and bearing conspicuous scar cells of trichoblasts, which often gave rise to adventitious branches (Fig. 9). The rhizoids were cut off from the posterior ends of pericentral cells and typically there was only one rhizoid per segment, often with digitate pads when totally developed (Fig. 9). Rhizoids were 30–60 (–80) µm in diameter and up to 800 (–1600) µm long. Erect axes grow from apical cells, which were 10–12.5 µm in diameter, the major axes reaching (50–) 60–110 (–130) µm in diameter; segments were (0.4–) 0.5–0.9 (–1.1) diameters long. Exogenous branches (i.e. those originated at the apices before the formation of the pericentral cells) were formed in the axils of trichoblasts (Figs 8, 12), mostly at regular intervals of six to eight (or nine) segments; they were arranged alternately (Figs 8, 11–12). Trichoblasts were usually numerous but sometimes scarcely developed and were borne (two or) three or four (or five) segments apart in a 1/4 spiral divergence; they were 12.5–17.5 µm in diameter at the base, pseudodichotomously branched up to three times (Fig. 8), deciduous, and left conspicuous scar cells. The branching pattern varied along the axes: exogenous branches arose from apices of erect axes every six to eight segments; later, the basal parts of erect axes developed rhizoids and became creeping; and then adventitious branches developed from some scar cells in these basal parts. Short prostrate axes arose from these adventitious branches, which had a prominent apical cell and lacked trichoblasts. Reproductive morphology: There was one tetrasporangium per segment. Long series of tetrasporangia were formed in a slight and irregular spiral containing up to 8 (–12) mature tetrasporangia. Series were frequently interrupted by segments lacking or with aborted tetrasporangia and, as a result, the tetrasporangia were scattered or formed short linear series of up to four (to six) mature ones. The tetrasporangia were ovate, (35–) 40–60 (–65) µm in diameter (Figs 18, 22). The gametophytes were dioecious. Procarps consisted of a four-celled carpogonial branch and two groups of sterile cells borne on the supporting cell (Fig. 19). Cystocarps were globular to ovoid when Polysiphonia foetidissima and P. schneideri in Europe 349 Figs 7–9. Polysiphonia foetidissima: vegetative morphology of Iberian specimens. 7. Habit of vegetative thallus. 8. Erect axis with trichoblasts three or four segments apart and branches seven or eight segments apart. 9. Creeping axis with rhizoids cut off from pericentral cells and two adventitious branches. Scale bars = 1 mm (Fig. 7) and 100 µm (Figs 8, 9). mature (Figs 20, 23) and (220–) 300–350 µm high and (160–) 210–285 (–300) µm in diameter, with an ostiole (75–) 100–150 (–170) µm in diameter. The carposporangia were clavate, (53–) 60–80 (–90) µm long and (18–) 20–25 (–30) µm wide. Spermatangial axes were located in the apical parts of erect main axes and branches (Figs 21, 24) and were borne three or four segments apart. Spermatangial axes were formed at one of the branches of the first dichotomy of fertile trichoblasts, the other branch persisting at maturity (Fig. 23). The spermatangial axes were cylindrical (Fig. 21), (100–) 110–170 (–188) µm long and (30–) 35–40 (–45) µm in diameter, with one to three sterile terminal cells when mature (Fig. 25). Occasionally, spermatangial axes were forked, as they were formed at the two branches of the first dichotomy of trichoblasts (Fig. 25). They consisted of a central axis with four pericentral cells P. Díaz-Tapia et al. 350 arising per axial cell. The pericentral cells bore a layer of quadrangular to elongate spermatangial mother cells of 12.5 × 10–12.5 µm, which developed elongate spermatia 10–12.5 × 5 µm. Polysiphonia foetidissima was collected throughout the year. Tetrasporangial thalli were frequently found year-round and they were observed in abundance in half of the collected samples. In contrast, gametophytes were more rarely observed: male and female structures were detected in less than 20% of the collections and only in April–June and September– October. Habitat and distribution In the Iberian Peninsula, P. foetidissima has been reported previously only in two Portuguese localities (Ardré, 1970). Our observations and those noted by Ardré show that P. foetidissima grows at moderately to extremely wave-exposed sites, usually on sandcovered rocks from the middle to the lower intertidal. It develops dense turfs over bare rock but it also grows over other turf-forming seaweeds like Rhodothamniella floridula, Polysiphonia nigra, Ophidocladus simpliciusculus or Pterosiphonia pennata. Interestingly, Polysiphonia foetidissima was occasionally found on subtidal maërl in a single collection from a maërl bed (at 10 m depth) in the Ría de Arousa (Galicia). Polysiphonia foetidissima was found in 28 locations throughout the Atlantic Iberian Peninsula (Fig. 26). Moreover, our examination of herbarium material from other regions confirms its occurrence at Stackpole Quay, Wales, U.K. (materials collected and identified by F. Bunker and C. Maggs in 1998); Atlantic France (Sant Vaast la Hogue: L 0796156; Biarritz: L 796154, L 796155, SANTAlgae 25433), and Atlantic North America (Bermuda: L 796144, MICH Phycotheca Boreali Americana, Algae of Bermuda N° 1890; Texas: MICH Port Isabella, channel side of jetty, 31. iii.1970). In addition, both molecular data and morphology support the conclusion that collections from Florida (U.S.A.) identified as N. tepida are conspecific with P. foetidissima. Figs 10–17. Polysiphonia foetidissima: vegetative morphology of Iberian specimens. 10. Entangled decumbent axes on a turf of Rhodothamniella floridula. 11, 12. Erect axes with trichoblasts 4 segments apart and branches 7 or 8 segments apart. 13–17. Cross- section of axes with 6–9 pericentral cells. Scale bars = 1 cm (Fig. 10), 400 µm (Fig. 11), 60 µm (Fig. 12) and 50 µm (Figs 13–17). Polysiphonia foetidissima and P. schneideri in Europe 351 Figs 18–21. Polysiphonia foetidissima: reproductive morphology of Iberian specimens. 18. Branch with tetrasporangia in spiral series. 19. Mature procarp showing the axial cell (ax), the supporting cell (su), one-celled second sterile group (st2) and four-celled carpogonial branch (1–4). 20. Erect axis with cystocarps. 21. Apical portion of an erect axis with cylindrical spermatangial branches. Scale bars = 100 µm (Figs 18, 20, 21), and 10 µm (Fig. 19). Polysiphonia schneideri Stuercke & Freshwater 2010, p. 302 HOLOTYPE: MICH (WNC-8782). TYPE LOCALITY: Wrightsville Beach, New Hanover County, North Carolina, USA. Description of material from Iberian Peninsula Vegetative morphology: The thalli were predominantly erect (Fig. 27), up to 5 cm long, and attached to the substratum by rhizoids that grew from the short, decumbent basal parts. Plants were red to purple. Rhizoids were cut off from pericentral cells (Fig. 28) and were abundant in the basal parts of axes, where there were often two per segment. Axes had six or seven pericentral cells (Figs 29, 30) and were ecorticate and (100–) 150–300 (–350) µm in diameter. Erect axes were pseudodichotomously or irregularly branched, up to seven orders. Branches formed in the axils of trichoblasts (Fig. 31), which were scarce, growing several segments apart, and irregularly arranged (Fig. 32). Reproductive morphology: Tetrasporangia were spherical, (75–) 80–100 µm in diameter, and P. Díaz-Tapia et al. 352 cylindrical to conical, (125–) 150–200 (–240) µm long and (25–) 35–45 (–60) µm in diameter, with several apical sterile cells (Fig. 36). Polysiphonia schneideri was collected in February and June, when tetrasporophytes, male and female gametophytes were found. Habitat and distribution Polysiphonia schneideri was found as a fouling organism at two sites in southern Spain (Fig. 26). One site consisted of aquaculture structures placed in the intertidal of the Bay of Cádiz, where P. schneideri was found entangled with P. denudata. The other site consisted of floating structures in the harbour of Barbate, where P. schneideri was growing on Balanus sp. and ascidians, together with Neosiphonia harveyii, Antithamnionella ternifolia and Antithamnion cruciatum. Discussion (including comparative data on other Polysiphonia species) Figs 22–25. Polysiphonia foetidissima: reproductive morphology of Iberian specimens. 22. Branches with tetrasporangia in spiral series. 23. Erect axes with cystocarps. 24. Apical portions of erect axes with cylindrical spermatangial branches. 25. Forked spermatangial branch. Scale bars = 400 µm (Figs 22–24) and 50 µm (Fig. 25). formed at the apical parts of erect axes in straight series, distending branches when mature (Fig. 33). Gametophytes were dioecious. Procarps consisted of a four-celled carpogonial branch (Fig. 34) and two groups of sterile cells borne on the supporting cell. Cystocarps were globular to ovoid when mature (Fig. 35), 350–450 (–500) µm high and (270–) 300–460 (–500) µm in diameter, with a narrow ostiole. Carposporangia were clavate, (75–) 85–110 (–125) µm long and (30–) 35–45 (–50) µm wide. The spermatangial branches were borne several segments apart in the upper parts of erect axes and each developed as one of the two first branches of a trichoblast, the other branch of which persisted at maturity. Spermatangial branches were The material of P. foetidissima from the Atlantic coasts of the Iberian Peninsula shares all the main taxonomic features with the type material. This red alga is a poorly known species, first described from specimens collected in the U.K. (Bornet, 1892) but reported from a few locations at mid–low latitudes on both sides of the North Atlantic Ocean (see Fig. 37 and references therein). Polysiphonia foetidissima shows some distinctive morphological features rarely found in other Polysiphonia, namely trichoblasts three or four segments apart, and spermatangial branches that sometimes fork. Maggs & Hommersand (1993), after examination of the type material, reported that trichoblasts were apparently borne on every segment. However, our own observations of the same material indicate that the trichoblasts are actually separated by the three or four naked segments noted above. Falkenberg (1901) and Hollenberg (1942) considered that the position of trichoblasts is a diagnostic feature in Polysiphonia. Usually, a single segment intervenes between successive trichoblasts. However, a few species are characterized by the occurrence of two or more segments between successive trichoblasts, the same feature that we have observed in P. foetidissima. Interestingly, several authors have noted that, when trichoblasts are separated by naked segments, the tetrasporangia develop in straight series (Hommersand, 1963; Kapraun, 1977; Kim et al., 2000; Stuercke & Freshwater, 2008). However, this alleged correlation between traits does not apply to P. foetidissima because its tetrasporangia are arranged in spiral series. Based on the descriptions and taxonomic comments provided in the literature, Polysiphonia foetidissima is very similar to P. stuposa, ‘Neosiphonia’ tepida (which our data indicate is conspecific with Polysiphonia foetidissima and P. schneideri in Europe 353 Fig. 26. Distribution of Polysiphonia foetidissima (circles) and P. schneideri (squares) along the Atlantic coasts of the Iberian Peninsula. P. foetidissima: see further discussion below), P. kappannae, P. nizamuddinii and P. isogona. Furthermore, we consider that P. exilis, P. confusa, P. schneideri and certain small, creeping forms of P. brodiei are also remarkably similar. By comparison, the 15 other species of Polysiphonia with a number of pericentral cells similar to that in P. foetidissima are distinguished from it in at least one or more of the following diagnostic features: rhizoid anatomy (in open connection vs. cut off from pericentral cells), branch development (not associated with trichoblasts vs. in the axils of trichoblasts), and habit (erect vs. prostrate or decumbent). These are some of the features commonly used to discriminate the various species of Polysiphonia (Falkenberg, 1901; Hollenberg, 1942; Segi, 1951; Meñez, 1964; Kapraun, 1977; Womersley, 1979; Maggs & Hommersand, 1993; Kim & Lee, 1999; Stuercke & Freshwater, 2008). Polysiphonia stuposa is another poorly known species. Originally described for Dalmatia (Kützing, 1864), this red alga is thought to be restricted to the Mediterranean (Fig. 37 and references therein). Its separation from P. foetidissima has been questioned by some authors. Hauck (1885) was the first author who regarded P. foetidissima as synonymous with P. stuposa. Despite this, subsequently Bornet (1892) validated the name P. foetidissima in Cocks’ exsiccate and considered that both species differed in the number of pericentral cells. However, various later authors have followed Hauck’s opinion and reported P. stuposa as synonymous with P. foetidissima (De Toni, 1903; Batten, 1923; Newton, 1931; Ardré, 1970). Maggs and Hommersand (1993) did not reach a definitive conclusion about the status of P. stuposa and P. foetidissima but noted that the type material of these two species appeared to be very similar. They retained the name P. foetidissima for the collection from the U.K. but pointed out that the name P. stuposa predates the probably conspecific P. foetidissima. More recently, Gómez-Garreta et al. (2001) have considered P. foetidissima to be a synonym of P. stuposa and grouped all the Mediterranean records for the two species under this name (Fig. 37). According to the literature, the only distinctive characters of P. stuposa are the 6–8 pericentral cells, the absence of trichoblasts, and the occurrence of segments 1–2 diameters long (Kützing, 1864). The absence of trichoblasts distinguishes this species from P. foetidissima, although several authors have argued that the abundance of trichoblasts is variable in several species of Polysiphonia (Hollenberg, 1942, 1968; Guimarães et al., 2004; Stuercke & Freshwater, 2008). Our detailed study of the type material of P. stuposa (Figs 38–42; holotype: MEL 2324435 and a fragment of this L0056078; isotype: L 0056079) and other collections from the Adriatic (L796145, 796147–796153, 796158) reveals that it can indeed be unambiguously distinguished from P. foetidissima, the main diagnostic feature being the origin of branches, which are not associated with trichoblasts in the case of P. stuposa (Fig. 40). This observation prevents us from considering these two species as synonymous and suggests that P. Díaz-Tapia et al. 354 Figs 27–36. Polysiphonia schneideri: vegetative and reproductive morphology of Iberian specimens. 27. Habit of vegetative thallus. 28. Rhizoids cut off from pericentral cells (arrow). 29, 30. Cross-sections of axes with 6 or 7 pericentral cells. 31. Branches growing in the axils of trichoblasts (arrow). 32. Trichoblasts borne several segments apart (arrowheads). 33. Tetrasporangia in straight series. 34. Procarp with four-celled carpogonial branch (1–4). 35. Mature cystocarp. 36. Spermatangial branch formed at one of the branches of the first dichotomy of a trichoblast. Scale bars = 0.5 cm (Fig. 27), 100 µm (Fig. 28), 50 µm (Figs 29–32, 36), 200 µm (Figs 33, 35) and 25 µm (Fig. 34). the range of P. stuposa, as well as the occurrence of P. foetidissima within the Mediterranean, requires further investigation. The transfer of Polysiphonia tepida to Neosiphonia by Guimarães et al. (2004) is questionable on the basis of our data. This transfer was based on observations of material from Brazil, which may have been misidentified because the specimens were reported to show traits (trichoblasts on every segment: see Oliveira Filho, 1969, pl 23, fig. 136; Guimarães et al., 2004) that do not match either our observations of the type material of P. tepida or the descriptions provided by other authors for collections from Atlantic North America (Hollenberg, 1958; Kapraun, 1977; Schneider & Searles, 1991, fig. 558; Dawes & Mathieson, 2008; Mamoozadeh & Freshwater, 2011). A similar confusion may also explain some recent reports of P. tepida in the Canary Islands (Rojas-González & AfonsoCarrillo, 2008). Instead, the morphological features of these plants from Brazil and the Canary Islands suggest that they could belong to a new species of Neosiphonia. Further research is warranted. True Polysiphonia tepida, reported from temperate coasts worldwide (Fig. 37 and references therein), was first described for North Carolina (Hollenberg, 1958). Its relationship with P. foetidissima has been rarely studied, even though these two species seemingly have overlapping ranges along the Atlantic coasts of North and Central America (Fig. 37). Hollenberg (1958) distinguished P. tepida from P. foetidissima because the latter (Bermuda material) had branches not arising in connection with trichoblasts. He also noted that the cortication at the base of some specimens of P. foetidissima from the U.K. reported by Newton (1931) differed from what he observed in P. tepida. Afterwards, only Taylor (1960) reported both P. foetidissima and P. tepida in the same work and it seems that he largely followed the criterion for Polysiphonia foetidissima and P. schneideri in Europe 355 Fig. 37. World distribution map of Polysiphonia foetidissima and related species. P. foetidissima (● type locality, ● reports with descriptions, ○ reports), P. isogona (▬ type locality, ▬ reports with descriptions), P. kappannae (♦ type locality), P. nizamudinii (▼ type locality), P. schneideri (* type locality), P. stuposa (■ type locality, □ reports) and P. tepida (▲ type locality, ▲ reports with descriptions, ∆ reports). References: 1, Kützing (1864); 2, Bornet (1892); 3, Collins & Hervey (1917); 4, Howe (1918); 5, Batten (1923); 6, Newton (1931); 7, Hollenberg (1958); 8, Lancelot (1966); 9, Sreenivasa Rao (1967); 10, Oliveira Filho (1969); 11, Hollenberg (1968); 12, Giaccone (1969); 13, Ardré (1970); 14, Cordeiro-Marino (1977); 15, Kapraun (1977); 16, Farooqui & Begum (1978); 17, Giaccone (1978); 18, Kapraun (1979); 19, Taylor (1960); 20, Schnetter & Schnetter (1981); 21, Weisscher (1983); 22, Audiffred & Prud´homme van Reine (1985); 23, Giaccone et al. (1985); 24, Conde & Soto (1986); 25, Silva et al. (1987); 26, Ballesteros (1990); 27, Adams (1991); 28, Schneider & Searles (1991); 29, Ballesteros (1993); 30, Lawson et al. (1995); 31, Silva et al. (1996); 32, Abbott (1999); 33, Coll & Oliveira (1999); 34, Rojas-González & Afonso-Carrillo (2000, 2008); 35, McDermid et al. (2002); 36, Womersley (2003); 37, Guimarães et al. (2004); 38, Suárez (2005); 39, Duncan & Lee Lum (2006); 40, Tsuda et al. (2006); 41, Dawes & Mathieson (2008); 42, Taskin et al. (2008); 43, Stuercke & Freshwater (2010); 44, Mamoozadeh & Freshwater (2011). separating the species that had originally been proposed by Hollenberg (1958). Unfortunately, Hollenberg did not study the type material of P. foetidissima, which, according to our observations as well as those provided by Maggs & Hommersand (1993), has branches formed in the axils of trichoblasts and is ecorticate throughout. These features are also found in the holotype of P. tepida (MICH, Herbarium of W.R. Taylor, collected by H.L. Blomquist in 1940 in Beaufort, North Carolina, U.S.A.; Figs 43–47) which is characterized by a predominantly prostrate habit (Fig. 43), axes with seven or eight pericentral cells (Fig. 44), rhizoids cut off from the pericentral cells (Fig. 45), and abundant trichoblasts separated by three or four naked segments (Fig. 47; for a more detailed comparative analysis see Table 1). The similarity between P. foetidissima and P. tepida is likewise consistent with our rbcL data, since the sequence available in GenBank (HM573552, labelled there and in our Fig. 1 as Neosiphonia tepida), produced by Mamoozadeh & Freshwater (2011) for P. tepida from Florida, showed only a very small divergence (< 0.4%) from our 12 collections of P. foetidissima from the Atlantic Iberian Peninsula. Therefore, we are compelled to propose the taxonomic synonymy of these two species, with P. foetidissima having nomenclatural priority. Polysiphonia kappannae and P. nizamuddinii were described from India (Sreenivasa Rao, 1967) and Pakistan (Farooqui & Begum, 1978), respectively (Fig. 37), but no additional reports have been provided after their original descriptions. They are very similar to one another as well as to P. foetidissima (Sreenivasa Rao, 1967; Kapraun, 1977) and the detailed descriptions provided by Sreenivasa Rao (1967) and Farooqui & Begum (1978) indicate that they can only be distinguished from P. foetidissima by the linear arrangement of tetrasporangia. However, the tetrasporangia in P. foetidissima are arranged in slight and irregular spiral series and sometimes appear to P. Díaz-Tapia et al. 356 Figs 38–42. Polysiphonia stuposa: type material. 38. Holotype (MEL 2324435, collected by Sandri in Dalmatia, Croatia). 39. Prostrate axis with rhizoids cut off from pericentral cells. 40. Apex of an erect axis showing branch origin not associated with trichoblasts. 41. Cross section of an axis with 7 pericentral cells. 42. Erect axes. Scale bars = 1 cm (Fig. 38), 150 µm (Fig. 39), 30 µm (Fig. 40), 20 µm (Fig. 41) and 600 µm (Fig. 42). form short straight series. Despite our efforts, we did not have the opportunity to examine type material of either species. However, considering the agreement in other features, we suggest that P. kappannae and P. nizamuddinii may be synonymous with P. foetidissima. Polysiphonia exilis, originally described by Harvey (1853) from Key West (Florida) and subsequently recorded from other locations (Table 1), shares several features with P. foetidissima (Table 1). However, their conspecificity seems unlikely according to our observations. We have examined the type material of P. exilis (TCD0012730, Figs 48–55) finding that the number of pericentral cells (nine; see Fig. 50) is larger than is usually observed in P. foetidissima. Likewise, the occurrence of trichoblasts on every segment (Figs 52, 53) and the irregular branching pattern that often results in secund series (Fig. 49) are other features that clearly differ from what is observed in P. foetidissima. Moreover, branches in P. exilis might not form at the apices since we did not observe any branches close to the tips. Conversely, we found numerous adventitious branches growing from scar cells of trichoblasts along the erect axes (Figs 53, 54). In comparison, P. foetidissima has exogenous branches and while it also shows branches growing from scar cells, the latter are restricted to prostrate axes or to the basal parts of the erect ones. Finally, and although not observed in the type material, the spermatangial axes of P. exilis lack sterile apical cells (Kapraun & Norris, 1982; Abbott, 1999). Polysiphonia confusa is another species morphologically similar to P. foetidissima (see Table 1). This Polysiphonia was originally described from California (Hollenberg, 1961) and seems to be restricted to the Pacific coasts of America. As with P. exilis, the main feature separating P. confusa from P. foetidissima is the occurrence of trichoblasts on every Figs 43–47. Polysiphonia tepida: type material. 43. Holotype (MICH, Herbarium of W.R. Taylor, collected by H.L. Blomquist in 1940 in Beaufort, North Carolina, U.S.A.). 44. Cross-section of an axis with eight pericentral cells. 45. Creeping axis with a rhizoid cut off from a pericentral cell. 46. Apical portions of an erect axis with abundant trichoblasts borne 3 or 4 segments apart and branches arising from the axils of trichoblasts. 47. Scar cell of a trichoblast on an erect axis. Scale bars = 2 cm (Fig. 43) and 50 µm (Figs 44–47). P. foetidissima P. schneideri P. brodiei P. confusa P. exilis P. isogona P. kappannae P. nizamuddinii P. stuposa P. tepida Type locality Plymouth, Cornwall, UK Wrightsville Beach, North Carolina, USA Bantry Bay, Cork, UK California, USA Key West, Florida Blind Bay, New Zealand Okga Port, India Paradisepoint, Pakistan Dalmatia, Croatia Beaufort, North Carolina USA Distribution Atlantic Europe and North America West Atlantic, southern Spain North Atlantic, Mediterranean, Pacific North America, Australia, New Zealand Pacific America Bermuda Caribbean Brazil, Australia Pacific islands, Maldives New Zealand, Southern Australia India Pakistan Mediterranean Sea Atlantic North America Pericentral cells (6 or) 7or 8 (or 9) (5 or) 6 (or 7) (6 or) 7 or 8 8–10 8–11 (7–) 9 or 10 (–12) 7–9 (8 or) 9 (or 10) 7 or8 (6 or) 7 or 8 Cortication ecorticate ecorticate slight to heavy ecorticate ecorticate ecorticate ecorticate ecorticate ecorticate ecorticate Branch origin axil of trichoblast axil of trichoblast axil of trichoblast axil of trichoblast not associated with trichoblast axil of trichoblast axil of trichoblast axil of trichoblast not associated with trichoblast axil of trichoblast Trichoblasts (abundance; segments between successive; divergence) abundant; 3–5; 1/4 few; not one per segment; irregular abundant; one per segment; 1/7– 1/8 abundant; one per segment; 1/4 abundant; one per segment; 1/4 variable abundant; several; 1/4 abundant; 3 or 4; – absent abundant to scarce; 3–5; 1/4 Carpogonial branch cells; Cystocarp 4; globose 4; globose 4; globose – – –; subspherical –; spherical –; globose Spermatangial branches basal branch of trichoblasts, sometimes forked basal branch of trichoblasts basal branch of trichoblasts – basal branch of trichoblasts or replacing them basal branch of trichoblasts Tetrasporangia spiral series linear series spiral series spiral series spiral series spiral series References This study Stuercke & Freshwater (2010); this study Maggs & Hommersand (1993); this study Hollenberg (1944 as P. inconspicua Hollenberg nom. illeg.; 1961) Harvey (1853); Hollenberg (1968); Kapraun & Norris (1982); Schneider & Searles (1997); Abbott (1999); this study Adams (1991); Womersley (2003). –; ovate to urceolate basal branch of trichoblasts – basal branch of trichoblasts, sometimes forked linear series linear series – spiral series Sreenivasa Rao (1967). Farooqui & Begum (1978). – Polysiphonia foetidissima and P. schneideri in Europe Table 1. Main taxonomic features of Polysiphonia foetidissima and similar species with 6–11 pericentral cells, rhizoids cut off from pericentral cells, and prostrate or creeping habit. Kützing (1864); this study Hollenberg (1958), Kapraun (1977), this work. 357 P. Díaz-Tapia et al. 358 Figs 48–55. Polysiphonia exilis: type material. 48. Holotype (TCD 0012730, collected by Harvey at Key West, Florida). 49. Erect axes with secund branches. 50. Cross-section of thallus with nine pericentral cells. 51. Rhizoid cut off from a pericentral cell (arrow). 52. Detail of an erect axis with scar cells of trichoblasts on every segment (arrows) and pericentral cells in straight rows. 53. Detail of an erect axis with young cicatrigenous branches growing from scar cells (arrowheads); the arrow shows a scar cell of a trichoblast. 54. Detail of an axis bearing a branch with young tetrasporangia forming a spiral series; the arrowhead indicates a young cicatrigenous branch. 55. Detail of a prostrate axis bearing erect axes at irregular intervals. Scale bars = 2.5 cm (Fig. 48), 3 mm (Fig. 49), 50 µm (Figs 50–53), 250 µm (Fig. 54) and 650 µm (Fig. 55). segment. Other distinctive features are: (1) a lower number of pericentral cells in P. foetidissima (7 or 8 vs. 8–10 in P. confusa), and (2) branches arising six to eight segments apart in P. foetidissima (at irregular intervals in P. confusa: see Hollenberg, 1961; Abbott & Hollenberg, 1976; Hollenberg & Norris, 1977). Our rbcL data indicate that P. isogona and P. foetidissima are close phylogenetically and could even be regarded as sister species. Actually, the sequence divergence found in this study (1.8%) falls at the upper end of the values reported for intraspecific rbcL variability for other Polysiphonia (Mamoozadeh & Freshwater, 2011). However, it seems unlikely that these two entities should be regarded as conspecific. Our results indicate intraspecific divergence could be very small within P. foetidissima. Variation between our 12 collections from the Iberian Peninsula and the sequence produced for material collected in Florida (by Mamoozadeh & Freshwater, 2011, as N. tepida;) is clearly lower (< 0.4%) than the separation between P. foetidissima and P. isogona. Furthermore, there are differences in some morphological traits, since P. isogona has (8 or) 9 or 10 (or up to 12) pericentral cells and wider axes (250– 300 µm in diameter) than P. foetidissima (Adams, 1991; Womersley, 2003). Furthermore, according to Adams (1991), P. isogona has trichoblasts borne on every segment (but see Womersley, 2003, for a different interpretation of their arrangement). Polysiphonia schneideri was recently described from Atlantic North America and Bermuda (Stuercke & Freshwater, 2010). It differs from P. foetidissima mostly in its six or seven pericentral cells, its predominantly erect habit, and its tetrasporangia arranged in straight series (Stuercke & Freshwater, 2010). Ours is the first record of P. schneideri in Europe and also the first report outside North America. The identity of the European collections is supported by our molecular data since the divergence estimated for samples collected on both sides of the Atlantic (0.1–0.8%) falls within the range of the values typically reported for intraspecific variation. Moreover, this variation compares well with values reported for collections of P. schneideri from the western Atlantic (0.41–0.66%) by Stuercke & Freshwater (2010). The morphological characteristics we observed fit well with the original description of the species. However, the usual number of pericentral cells in our collections is seven, while in the western Atlantic six pericentral cells are most commonly reported. Polysiphonia schneideri was collected at two sites from the province of Cádiz Polysiphonia foetidissima and P. schneideri in Europe 359 Figs 56–61. Polysiphonia brodiei: small and creeping forms in the Iberian Peninsula. 56. Habit of a tetrasporophyte growing on Codium adhaerens. 57. Spermatangial branches borne on every segment and without sterile apical cells. 58. Erect axes with scar cells of trichoblasts on every segment. 59–61. Upper portions of erect axes with branches arising in the axils of trichoblasts, 3 or 4 segments apart. Scale bars = 2 cm (Fig. 56), 80 µm (Fig. 57), 50 µm (Figs 58, 59), 500 µm (Fig. 60) and 200 µm (Fig. 61). (Andalusia, southern Spain, Fig. 5) and was always associated with man-made structures, namely floating docks and artificial substrata for aquaculture (mostly for Crassostrea gigas or Ruditapes decussatus, but also several species of fishes). Man-made structures are commonly considered typical habitats for algal invaders (McIvor et al., 2001) and since it seems absent from pristine sites nearby, we believe P. schneideri is a new introduction to Europe. The occurrence of this species in Cádiz could result from importation of molluscs or from ship fouling, which are commonly reported vectors of seaweeds (Mineur et al., 2007a, b). However, Polysiphonia includes a large number of species, which are variable in morphology, difficult to identify, and sometimes cryptic (McIvor et al., 2001; Stuercke & Freshwater, 2010; Geoffroy et al., 2012). Thus, it is difficult to know if P. schneideri is a new addition to the European exotic flora or if it is an overlooked native species. It is worth noting that, even in the western Atlantic, P. schneideri is also commonly P. Díaz-Tapia et al. found on artificial substrates (Stuercke & Freshwater, 2010; Mamoozadeh & Freshwater, 2012). The last species to be considered here is P. brodiei. Typically, P. brodiei is erect and heavily corticated (Maggs & Hommersand, 1993). However, it is extremely variable across habitats and seasons and certain small, creeping forms of P. brodiei that are slightly corticated to ecorticate (Fig. 56) can be misidentified as P. foetidissima (Table 1). However, our molecular results confirm the conspecificity of the small, creeping forms with other collections of P. brodiei (Fig. 1), despite their distinctive habit. They can be distinguished from P. foetidissima by the presence of trichoblasts and scar cells on every segment in P. brodiei (Fig. 58), so that P. brodiei has spermatangial branches on all segments (Fig. 57), whereas P. foetidissima has spermatangial branches three or four segments apart. Additional features of P. brodiei useful to distinguish it from P. foetidissima are that P. brodiei lacks sterile apical cells in the spermatangial branches (Fig. 57) and has branches arising from the erect axes three or four segments (or trichoblasts) apart (Figs 59– 61). Along the Atlantic coast of the Iberian Peninsula we have found that these small, creeping forms usually grow on Codium adhaerens. Interestingly, Batten (1923) and Newton (1931) noted that material from the U.K. labelled as P. foetidissima sometimes showed cortication at the base. This feature matches our observations in creeping forms of P. brodiei and, since part of the material studied by Batten (1923) had been collected on Codium adhaerens, they could represent misidentified collections of P. brodiei. Unfortunately, their exact identity cannot be confirmed because these reports do not appear to be supported by voucher specimens (Maggs & Hommersand, 1993). As mentioned in the Introduction, Polysiphonia, is one of the largest genera within Rhodophyta, with approximately 200 species (Guiry & Guiry, 2012; Mamoozadeh & Freshwater, 2012). Recently, Neosiphonia has been separated from Polysiphonia and 30 species have been transferred to the new genus, which is characterized, among others, by having three-celled carpogonial branches (Kim & Lee, 1999; Guiry & Guiry, 2012). This trait prevents us from considering that Polysiphonia foetidissima and P. schneideri, which have four-celled carpogonial branches, could be better accommodated in the genus Neosiphonia. Acknowledgements We thank M.J. Wynne for critically reading an early version of the manuscript and sending collections from the herbarium of Michigan; B. De Reviers and L. Le Gall for their attendance during the stay at PC; the curators of Leiden, Melbourne, Trinity College of Dublin and Santiago de Compostela herbaria for lending collections 360 of Polysiphonia spp.; M. Fernández-Lora for providing material of P. schneideri from Santa Leocadia (Cádiz); J. Afonso-Carrillo for providing material of ‘P. tepida’ from the Canary Islands, V. Peña for providing material of P. foetidissima from maërl beds, and C. Maggs and F. Bunker for providing material of P. foetidissima from the U.K.; and I. Maneiro and L. Couceiro for their assistance with the molecular analysis; and two anonymous reviewers for their suggestions and comments on the manuscript. This study was funded by a grant from Xunta de Galicia and supported by the project CGL2009-09495/BOS (Ministerio de Ciencia e Innovación, partially funded by FEDER). It was also supported by National Research Foundation of Korea Grant funded by the Korean Government (20110003792) to M.S. Kim. Supplementary information The following supplementary material is available for this article, accessible via the Supplementary Content tab on the article’s online page at Table S1. 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