Molecular data confirm the existence of attached crustose tetrasporangial thalli in Phymatolithon calcareum (Melobesioideae, Hapalidiaceae, Rhodophyta) from the Mediterranean Sea

Aquatic Botany 134 (2016) 75–81

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Short communication

Molecular data confirm the existence of attached crustose tetrasporangial thalli in Phymatolithon calcareum (Melobesioideae, Hapalidiaceae, Rhodophyta) from the Mediterranean Sea Marion A. Wolf a,∗ , Annalisa Falace b,∗ , Sara Kaleb b , Isabella Moro a a b

Department of Biology, University of Padova, via Ugo Bassi 58/B, 35121 Padova, Italy Department of Life Science, University of Trieste, Via L. Giorgieri 10, 34127 Trieste, Italy

a r t i c l e

i n f o

Article history: Received 2 December 2015 Received in revised form 8 June 2016 Accepted 14 July 2016 Available online 15 July 2016 Keywords: Maërl Non-geniculate coralline red algae nSSU psbA Rhodolith

a b s t r a c t Phymatolithon calcareum is a species of non-geniculate coralline red algae that commonly forms maërl beds. Tetrasporangial thalli of this species are characterized by unattached growths (rhodoliths), which develop as a branched system without a central nodule, while the attached crustose forms are uncommon and are known only from Brittany (Atlantic coasts of France). In this study we confirm with molecular data the existence of attached crustose tetrasporangial thalli and describe for the first time epilithic, fruticose tetrasporangial specimens from the Mediterranean Sea (Vis Island, Croatia). Since the identification of coralline algal species is difficult due to the high morphological plasticity of the group, molecular analyses based on the nuclear nSSU and the plastidial psbA markers were necessary to correctly identify the specimens. Our study shows that P. calcareum tetrasporangial thalli have the ability to grow both unattached and attached, and we argue this to be in relation to the environmental conditions found at different depths. This ability permits the alga to colonize different habitats of the coastal environment. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Non-geniculate coralline red algae (Corallinales, Hapalidiales, Sporolithales) include attached crustose and free-living morphologies. Free-living specimens, commonly known as rhodoliths, form extensive beds worldwide in the euphotic zone and constitute one of the most important marine habitats for the establishment of many associated macroalgae and invertebrates (Birkett et al., 1998; ˜ and Barbara, 2008; Pereira-Filho et al., 2012; Foster, 2001; Pena Villas-Bôas et al., 2015). Consequently these environments have a high conservation status in European legislation through the Habi˜ and Barbara, 2008; Basso et al., tats Directive (92/43/EEC) (Pena 2015). Along the Atlantic European coasts rhodolith beds, known by the Breton name maërl, are mostly composed of Lithothamnion corallioides (P.Crouan & H.Crouan) P.Crouan & H.Crouan and Phy˜ and matolithon calcareum (Pallas) W.H.Adey & D.L.McKibbin (Pena Bárbara, 2008). Here, the tetrasporangial thalli of P. calcareum are represented almost entirely by the rhodolith form.

∗ Corresponding authors. E-mail addresses: [email protected] (M.A. Wolf), [email protected] (A. Falace). http://dx.doi.org/10.1016/j.aquabot.2016.07.006 0304-3770/© 2016 Elsevier B.V. All rights reserved.

Cabioch (1966, 1970) and Mendoza and Cabioch (1998) gave an account of small basal crusts bearing tetrasporangial conceptacles in maërl beds from Brittany (France), while Cabioch (1970), ˜ et al. (2014) reported thin Mendoza and Cabioch (1998) and Pena attached gametophytes growing on gravel, dead maërl and pebbles also from the same area. According to the literature, thick crustose tetrasporangial thalli attached to hard bottoms were never described for this species (Chamberlain and Irvine, 1994; Bressan ˜ and Bárbara, 2004, 2008; Hernándezand Babbini 2003; Pena ˜ et al., 2014; Hernández-Kantún et al., Kantún et al., 2014; Pena 2015). Recently, Hernández-Kantún et al. (2015) suggested that in rhodolith-forming species, like P. calcareum, the free-living form appears to be a synapomorphy in tetrasporangial plants, suggesting a complete adaptation to the unattached habit. The identification of coralline red algae based solely on morphological and anatomical features is complicated because of the high morphological plasticity in these algae (Steneck, 1986; Woelkerling, 1988). Morphological variations in both unattached (Foster, 2001; Berlandi et al., 2012; Pereira-Filho et al., 2012) and attached (Woelkerling et al., 1993; Figueiredo and Steneck, 2000; McCoy and Kamenos, 2015) forms can be observed even within a single species when influenced by local hydrodynamic conditions ˜ and Bárbara, 2009) or by factors not related to and depth (Pena water motion (Steller and Foster, 1995).

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It is particularly for this reason (morphological plasticity) that DNA sequencing is proving to be invaluable in identifying specimens of non-geniculate coralline red algae, where morphological and anatomical analyses are not reliable for separating taxa (Pardo et al., 2014). In this context, the employment of DNA barcode technique led to the positive identification of cryptic non-geniculate coralline red algal specimens (Pardo et al., 2014). Here we confirm the existence of attached tetrasporangial thalli and report for the first time crustose forms in tetrasporangial specimens of P. calcareum from the Mediterranean Sea (Vis Island, Croatia). 2. Methods 2.1. Sampling The specimens were scraped from calcareous substrata with a hammer and chisel by SCUBA diving in August 2008 in an area of Vanjska Sika, Vis Island (Croatia, 43.008◦ N; 16.0643◦ E) at a depth of about 20 m. 2.2. Morphological and anatomical analyses Specimens were air-dried and placed in silica gel. Only scanning electron microscopy was undertaken. Fragments of material were mounted on aluminum stubs with acrylic adhesive and then coated with gold/palladium in an Edwards S150 Sputter Coater (Crawley, UK) prior to viewing with a LEICA Steroscan 430i scanning electron microscope (Cambridge, UK) at an accelerated voltage of 20 kV. Anatomical terminology follows Woelkerling (1988) and growth forms terminology follows Woelkerling et al. (1993). The samples were deposited in the Herbarium TSB (Università degli Studi di Trieste, Trieste, Italy) as vouchers TSB-PHM CLC 3a and TSB-PHM CLC 3b. 2.3. Molecular analyses Specimens were identified based on the nuclear smallsubunit ribosomal RNA (nSSU) and the plastidial gene coding for the D1 protein of photosystem II (psbA). Total genomic DNA was purified following Broom et al. (2008) and the partial nSSU region was amplified and sequenced as reported in Wolf et al. (2015). For amplification and sequencing of the plastidial psbA gene two specific primers were constructed: Hyd psbA F 5 -ATTCATCATTGCWTTYGTT-3 and Hyd psbA R 5 ARTTGAATGTACCAGATATACC-3 . Final consensus sequences were assembled using the SeqMan II program from the Lasergene software package (DNAStar©, Madison, WI). Identity of new sequences was checked by using the BLAST program (Altschul et al., 1990) available at the USA National Center for Biotechnology Information (NCBI) web server (http://www.ncbi.nlm.nih.gov), and the sequence alignment was obtained by the ClustalW computer program (Thompson et al., 1994). The nSSU and the psbA sequences of P. calcareum (TSB-PHM CLC 3a) were deposited in GenBank with the following accession numbers: LN913084 and LT221117 respectively. To infer the phylogenetic position of our specimen a dataset formed by the concatenate sequences of the two genes was constructed. The dataset comprised both our nSSU + psbA sequences and sequences of taxa with the highest identity found through the BLAST search (5 specimens belonging to the genus Phymatolithon and 2 sequences of Lithothamnion chosen as outgroup). The dataset also included the sequences of the neotype of P. calcareum carried out in a study by Hernández-Kantún et al. (2015). The phylogenetic reconstruction was obtained using the Neighbor Joining (NJ) method (Saitou and Nei, 1987) with the MEGA 5.1 (Molecular Evolutionary Genetics Analysis) program (Tamura et al., 2011) by applying the T92 model of evolution (Tamura, 1992).

The nSSU + psbA dataset had 124 variable sites and 66 parsimony informative sites on 932 aligned positions. 3. Observations Phymatolithon calcareum (Pallas) W.H.Adey & D.L.McKibbin, 1970: 100 Figs. 1(A–D) 2(A–E) Basionym: Millepora calcarea Pallas, 1766: 265 Neotype: W.F. Farnham; 11.xii.1983; subtidal. BM Box 1626 (Chamberlain and Irvine 1994: 212). See Woelkerling and Irvine (1986: Figs. 1–15) for more information on the neotype specimen. Isoneotypes: BM, LTBType locality: Falmouth Harbour, Cornwall, England (Chamberlain and Irvine 1994: 212). Homotypic Synonyms: Nullipora calcareum (Pallas) Lamarck 1801: 574; Melobesia calcarea (Pallas) Harvey 1849: 110; Spongites calcareus (Pallas) Kützing 1849: 699; Lithothamnion calcareum (Pallas) Areschoug in J.Areschoug 1852: 523; Lithophyllum calcareum (Pallas) Foslie 1898: 9; Paraspora calcarea (Pallas) Heydrich 1908: 55. Heterotypic Synonyms: Lithothamnion corallioides f. subsimplex Batters 1892: 177; Phymatolithon polymorphum Foslie 1898: 4; Lithothamnion calcareum f. subsimplex (Batters) Foslie 1900: 13; Eleutherospora polymorpha (Linnaeus) Heydrich 1900: 65; Lithothamnion hamelii Me. Lemoine 1931: 9. Etymology: adjective (Latin), chalky, limy (Stearn, 1973). The epithet presumably makes reference to the calcareous nature of the unattached specimens originally described by Pallas to be “composed of a hard, smooth white surface” (Woelkerling and Irvine 1986: 56). 3.1. Habit Specimens grew attached to other calcareous substrata on a vertical cliff. Thalli were lumpy to fruticose with protuberances up to 2 cm high (Fig. 1A). Protuberances were claviform ending in rounded tips (Fig. 1A, B). 3.2. Vegetative and reproductive anatomy The thallus was dorsiventrally organised, thin and monomerous, comprising a core of 5–6 layers of non-coaxial prostrate filaments (20–40 ␮m thick) running more or less parallel to the substratum. These filaments comprised rectangular cells that measured 8–11 ␮m long and 2–5 ␮m in diameter. Prostrate filaments curved upward to form a peripheral region (to 700 ␮m thick). The core region of the vegetative thallus consisted of less than 10% of the thallus thickness (Fig. 1C). Peripheral filaments were composed of rounded/ovoid to rectangular cells (3–10 ␮m long and 3–6 ␮m in diameter). Cell fusions were abundant in both prostrate and peripheral filaments (Fig. 1D); no secondary pit connections were observed. In surface view, the epithallial cells were polygonal with thickened calcareous ridges. In vertical section there were one or two layers of flattened to domed epithallial cells (1–3 ␮m long and 3–5 ␮m in diameter) subtended by initials (2–5 ␮m long and 4–6 ␮m in diameter) that were as short as or shorter than their immediate inward derivatives (Fig. 1D). Protuberances developed from localised proliferations of peripheral filaments. In longitudinal sections protuberances were up to 2000 ␮m thick (Fig. 2A), consisting of arching tiers of longitudinal filaments composed of rounded/ovoid to rectangular cells (3–9 ␮m long and 3–5 ␮m in diameter). Trichochytes were not observed. Specimens bore multiporate conceptacles (Figs. 2B, 2C) that measured up to 390 ␮m externally, were slightly sunken to slightly raised above the surrounding thallus surface, and which bore rims up to 70 ␮m wide (Fig. 2C). Conceptacle pore plates were concave to flat with up to

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Fig. 1. Vegetative anatomy of attached, tetrasporangial P. calcareum (TSB-PHM CLC 3a). A. Attached thallus with lumpy to fruticose growth form; protuberances ending in rounded tips (arrowheads). B. Magnified view of tetrasporangial conceptacles scattered across the protuberances. C. Vertical fracture through the thallus showing a thin core (c) and a thick peripheral (p) region. D. Vertical fracture of the outer thallus showing flattened to domed epithallial cells (e), short subepithallial initials (i) and cell fusions (f) between contiguous cells of peripheral filaments. Scale bars = 15 mm (A), 5 mm (B), 100 ␮m (C), 10 ␮m (D).

60 pores. Pore openings were bordered by 5–7 (mostly 6) rosette cells that were similar in shape and size to other epithallial cells in the pore plate (Fig. 2D). In vertical section, the conceptacle roof was composed of 4–5 layers of cells with what appear to be elongated epithallial cells (4–7 ␮m long and 3–4 ␮m in diameter) (Fig. 2E). Conceptacle chambers were elliptical and measured 220–310 ␮m in diameter and 68–115 ␮m in height. Examined conceptacles were senescent and devoid of contents but they were interpreted as tetrasporangial since in P. calcareum multiporate conceptacles are currently known to be mostly tetrasporangial (Adey and McKibbin, 1970; Woelkerling and Irvine, 1986; Chamberlain and ˜ and Bárbara, 2004; Irvine, 1994; Mendoza and Cabioch, 1998; Pena Hernández-Kantún et al., 2015), as opposed to bisporangial. Buried conceptacles were frequently observed in vertical sections (Fig. 2A). A comparative summary of data related to tetrasporangial conceptacle features in P. calcareum from different geographical areas is given in Table 1. 3.3. Specimens examined Vis Island, Eastern Mediterranean (Croatia, 43.008◦ N; 16.0643◦ E), S. Kaleb, 20 m, 3 August 2008, TSB-PHM CLC 3a; Vis Island, Eastern Mediterranean (Croatia, 43.008◦ N; 16.0643◦ E), S. Kaleb, 20 m, 3 August 2008, TSB-PHM CLC3b. 3.4. Molecular analyses The phylogenetic reconstruction based on the nSSU + psbA multiple alignment and obtained from the NJ analysis is shown in

Fig. 3. The sequences of the Lithothamnion species formed a well-supported clade basal to that of the Phymatolithon ones. The concatenate sequences of our Mediterranean specimen were placed in this latter clade and clustered with the sequences of the neotype of P. calcareum (100% bootstrap value), collected by W.F. Farnham in 1983 and conserved at the Natural History Museum, London (Herbarium number: BM000712373). The sequence divergence between the two specimens was 0%. The molecular results therefore confirm that our sample is P. calcareum despite the habit differences between our Adriatic specimen and the neotype specimen reported in Woelkerling and Irvine (1986). 3.5. Distribution Phymatolithon calcareum is common and widely distributed along the Atlantic coasts of Europe including Norway (Pardo et al., 2014), Sweden (Suneson, 1958), France (Lemoine, 1910; Cabioch, ˜ 1966, 1969; Mendoza and Cabioch, 1998; Pardo et al., 2014; Pena ˜ and Barbara, et al., 2014), Spain (Adey and McKibbin, 1970; Pena ˜ et al., 2014), United Kingdom 2004, 2008; Pardo et al., 2014; Pena (Adey and Adey, 1973; Woelkerling and Irvine, 1986; Chamberlain ˜ et al., 2014), Ireland (Adey and Adey, 1973; and Irvine, 1994; Pena Bosence, 1976) and the Azores (Rosas-Alquicira et al., 2009). Westward, along the Northern Atlantic coasts, it was found in the Gulf of Maine (Adey, 1966). It has been reported also from the eastern and western Mediterranean (Babbini and Bressan, 1997; Bressan and Babbini, 2003; present work). P. calcareum has also been described for Alaska (Konar et al., 2006). According to Wilks and Woelkerling (1994) records

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Fig. 2. Reproductive anatomy of attached, tetrasporangial P. calcareum (TSB-PHM CLC 3a). A. Vertical fracture through a protuberance showing buried tetrasporangial conceptacles. B. Magnified view of a protuberance showing densely aggregated tetrasporangial conceptacles. C. Single multiporate tetrasporangial conceptacle with slightly raised rim and sunken pore plate. D. Magnified view of a tetrasporangial conceptacle pore plate showing pores (p) and rosette cells (*) surrounding the pores. E. Vertical fracture of a tetrasporangial conceptacle roof showing what appears to be elongated epithallial cells (arrowheads). Scale bars = 200 ␮m (A), 500 ␮m (B), 50 ␮m (C), 5 ␮m (D), 5 ␮m (E).

from southern hemisphere require verification nevertheless more recently it was described for the Brazilian coasts (Costa et al., 2014). 4. Discussion Phymatolithon calcareum is mainly known as a branched, fruticose, unattached species (see Chamberlain and Irvine, 1994) and is one of the most important components of European maërl beds (Hall-Spencer et al., 2010). Till now, attached forms were not known for the Mediterranean (Bressan and Babbini, 2003; Kaleb et al., 2012) and have only rarely been recorded in the literature from other locations, despite the fact that the type locality (Cornwall, England), other British Isles, as well as the Spanish Atlantic coasts were extensively studied (Adey and McKibbin, 1970; Farnham and Bishop, 1985; Chamberlain and Irvine, 1994; Hernández-Kantún ˜ et al., 2014; Hernández-Kantún et al., 2015). et al., 2014; Pena While for the Atlantic coasts, Cabioch (1966, 1970) and Mendoza and Cabioch (1998) reported small attached, tetrasporangial crusts

associated with maërl beds from Brittany (France), for the Mediterranean, Hernández-Kantún et al. (2015) alluded to the presence of both rhodolith and attached tetrasporangial crusts based on a previous study by Bressan and Babbini (2003). The Hernández-Kantún et al. (2015) statement, however, was erroneously reported because the Bressan and Babbini (2003) work was based solely on previous European descriptions and on voucher specimens deposited at TSB (Università degli Studi di Trieste, Trieste, Italy), which incidentally does not contain any attached forms. The current study thus represents the first reporting of thick, fruticose tetrasporangial specimens found as attached crusts on vertical coralligenous cliffs from the Mediterranean Sea. Tetrasporangial conceptacle characters of the specimens from this study match those of the neotype material from the British Isles described by Woelkerling and Irvine (1986), and even more so those described for other British Isles specimens by Chamberlain and Irvine (1994). Ironically, the specimens that are geographically most closely associated (Adey and McKibbin, 1970; Mendoza and

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Table 1 Comparison of tetrasporangial conceptacle characters of Phymatolithon calcareum from different geographical areas. Central Adriatica

British Isles (neotype)b

British Islesc

Mediterraneand

Atlantic coast of Francee

Atlantic coast of Spainf

Disposition with respect to thallus surface External diameter Chamber diameter Chamber height Peripheral rim Peripheral rim thickness

Sunken or slightly raised

Sunken or slightly raised

Sunken or slightly raised

Sunken or slightly raised

Sunken or slightly raised

Sunken*

329–390 ␮m 220–310 ␮m 68–115 ␮m Yes 64–70

to 500 ␮m to 350 ␮m c.a.108* Yes/No ND

to 500 ␮m 230–350 ␮m 117–130 ␮m Yes/No ND

200–500 ␮m 120–150 ␮m 50–60 ␮m Yes ND

c.a. 220–320* 120–250 ␮m 50–60 ␮m Yes/No* ND

ND 126–190 ␮m 58–89 ␮m Yes* ND

Pore plate diameter No. of pores in roof Pore diameter No. of rosette cells

137–256 ␮m 50–60 4–8 ␮m 5–7 (mostly 6)

ND ND ND 6–7* (mostly 6)

ND 30–60 ND c.a. 6

ND 30–60 ND ND

ND ND ND 5–6*

54–126 ␮m ND ND ND

Roof thickness No. of cell layers in roof Elongated apparent epithallial cells in roof

20–30 ␮m 3–5 Yes

ND 3–5 Yes

30–40 ␮m 4–7* Yes*

ND ND ND

ND 3–5 ND

18–22 ␮m 4–5* No*

Old conceptacles buried Tetrasporangia length breadth

Yes

None seen

Yes

ND

ND

No

88–126 ␮m 49–64 ␮m

to 125 ␮m 60 ␮m

90–125 ␮m 49–73 ␮m

ND ND

50 ␮m 40–45 ␮m

ND ND

a b c d e f *

The present study. Woelkerling and Irvine (1986). Chamberlain and Irvine (1994). Bressan and Babbini (2003). Mendoza and Cabioch (1998). Adey and McKibbin (1970). ND: no data. Data derived from pictures/illustrations.

Fig. 3. NJ tree inferred from nSSU and psbA gene sequences calculated using the T92 model of evolution. Numbers near nodes indicate bootstrap values (> 50%). The Adriatic specimen sequences determined in this work are indicated as LN913084/LT221117 Phymatolithon sp. The type species for each genus is bolded.

Cabioch, 1998; Bressan and Babbini, 2003) are anatomically least similar (with generally smaller internal specifications) to our specimens (Table 1). We may have to question whether indeed any of these other specimens are molecularly identical to P. calcareum. Historically, the key features employed to delimit Phymatolithon species are related not only to the reproductive features, but also to habit, growth form and thickness of the core region compared to total thallus thickness (Chamberlain, 1991; Chamberlain and Irvine, ˜ et al., 2015). Based on our molecular 1994; Kaleb et al., 2012; Pena data that now confirms the presence of attached tetrasporangial forms of P. calcareum from the Mediterranean Sea, these features are inadequate for a correct identification of the species. Presum-

ably attached tetrasporangial thalli of P. calcareum may have been misidentified for other species (e.g. P. lenormandii (Areschoug) W.H.Adey, P. lamii (Me.Lemoine) Y.M.Chamberlain and P. laevigatum (Foslie) Foslie) of the genus that usually maintain the attached form in both gametangial and tetrasporangial thalli. Phymatolihon laevigatum, in particular, shares with P. calcareum a similar external shape of the tetrasporangial conceptacles and the thickness of the core region (Chamberlain and Irvine, 1994). In the absence of clear diagnostic features, the molecular analyses were essential for the identification of the attached tetrasporangial thalli of P. calcareum described in this study.

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While Hernández-Kantún et al. (2015) suggested that the rhodolith form in some coralline taxa, including tetrasporangial thalli of P. calcareum, can be considered a synapomorphy and a complete adaptation to the unattached habit, our study does not support this suggestion. Here we provide evidence that P. calcareum tetrasporangial thalli have the ability to grow both attached and unattached as rhodoliths. All attached specimens from this study were found growing epilithically at 20 m depth in association with Lithophyllum stictaeforme (Areschoug) Hauck while unattached, rhodolith forming specimens were found deeper at 50 m growing on sediments. Rhodolith forms have been reported to have strict habitat requirements (e.g. sediments with high calcareous content, moderate water movement, low anthropogenic disturbance) (Shepherd, 2013). If water motion, for example, is too high, the thalli can be rolled away or can be damaged by erosion; if water motion is too low, thalli can become buried by sediments (Foster, 2001). Moreover, hard substrata are not optimal habitats for rhodoliths (Foster, 2001) because, also in this case, thalli can easily be carried away and can be subject to abrasion and competition from attached organisms (Foster, 2001). On the other hand, in favorable growing conditions, rhodoliths can occur in high densities to form deposits of living and dead aggregations over large areas, and their skeletons can persist after death to form relic beds, beaches, and fossil deposits (Foster, 2001; Shepherd, 2013). All these considerations suggest that environmental conditions associated with depth play a strong role in the relative distributions of both attached and unattached forms. In conclusion, unattached rhodolith forms of P. calcareum are more abundant in environments that favour that form. Also, the unattached form does not represent a fixed synapomorphic character. The ability to grow both attached and unattached permits P. calcareum to colonize a greater diversity of habitats in response to different environmental conditions. Acknowledgment This work was supported by grants provided by the Scientific Research Grant (EX 60%) of the University of Padova, Italy. References Adey, W.H., 1966. The genera Lithothamnium, leptophytum (nov. gen.) and Phymatolithon in the Gulf of Maine. Hydrobiologia 28, 321–370. Adey, W.H., Adey, P.J., 1973. Studies on the biosystematics and ecology of epilithic crustose Corallinaceae of the British Isles. Br. Phycol. J. 8, 343–407. Adey, W.H., McKibbin, D., 1970. Studies on the maërl species Phymatolithon calcareum (Pallas) nov: comb. and Lithothamnion coralloides Crouan in the Ria de Vigo. Bot. Mar. 13, 100–106. Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local alignment search tool. J. Mol. Biol. 215, 403–410. Babbini, L., Bressan, G., 1997. Recensement de Corallinacées de la Mer Méditerranée et considérations phytogéographiques. Bibl. Phycol. 103, 1–421. Basso, D., Babbini, L., Kaleb, S., Bracchi, V.A., Falace, A., 2015. Monitoring deep Mediterranean rhodolith beds. Aquat. Conserv.: Mar. Freshw. Ecosyst., http:// dx.doi.org/10.1002/aqc.2586. Berlandi, R.M., Figueiredo, M.A.O., Paiva, P.C., 2012. Rhodolith morphology and the diversity of polychaetes off the southeastern Brazilian coast. J. Coast. Res. 28, 280–287. Birkett, D.A., Maggs, C.A., Dring, M.J., 1998. Maerl (volume V). An Overview of Dynamic and Sensitivity Characteristics for Conservation Management of Marine SACs. Scottish Association for Marine Science (UK Marine SACs Project). Bosence, D.W.J., 1976. Ecological studies on two unattached coralline algae from western Ireland. Palaeontology 19, 365–395. Bressan, G., Babbini, L., 2003. Biodiversità marina delle coste Italiane: corallinales del Mar Mediterraneo: guida alla determinazione. Biol. Mar. Medit. 10, 1–237. Broom, J.E.S., Hart, D.R., Farr, T.J., Nelson, W.A., Neill, K.F., Harvey, A.S., Woelkerling, W.J., 2008. Utility of psbA and nSSU for phylogenetic reconstruction in the Corallinales based on New Zealand taxa. Mol. Phyl. Evol. 46, 958–973. Cabioch, J., 1966. Contribution á l’Etude morphologique, anatomique et systematique de deux Melobesiées: Lithothamnion calcareum (Pallas) Areschoug et Lithothamnion coralloides Crouan. Bot. Mar. 9, 33–53. Cabioch, J., 1969. Les fonds de maerl de la baie de Morlaix et leur peuplement végétal. Cah. Biol. Mar. 10, 139–161.

Cabioch, J., 1970. Le Maërl Des Cótes De Bretagne Et La Problème De Sa Survie, 7. Penn ar Bed, pp. 421–429. Chamberlain, Y.M., Irvine, L.M., 1994. Seaweeds of the British Isles. 1 Rhodophyta Part 2B Corallinales, Hildenbrandiales. HMSO, London. Chamberlain, Y.M., 1991. Observations on Phymatolithon lamii (Lemoine) Y. Chamberlain comb. nov. (Rhodophyta, Corallinales) in the British Isles with an assessment of its relationship to P. rugulosum, Lithophyllum lamii and L. melobesioides. Brit. Phycol J. 26, 219–233. Costa, I.O., Horta, P.A., Bergstrom, E., de Castro Nunes, J.M., 2014. Taxonomic study of crustose coralline algae off the northeastern Brazilian coast. Phytotaxa 190 (1), 130–161. Farnham, W.F., Bishop, G., 1985. A survey of the fal estuary (Cornwall). Progr. Underw. Sci. 10, 53–63. Figueiredo, M.A.O., Steneck, R.S., 2000. Floristic and ecological studies of crustose coralline algae on Brazil’s Abrolhos reefs. In: Proceedings of the 9th International Coral Reef Symposium, Bali, pp. 493–498. Foster, M.S., 2001. Rhodoliths: between rocks and soft places. J. Phycol. 37, 659–667. Hall-Spencer, J.M., Kelly, J., Maggs, C.A., 2010. Background document for maërl beds. In: Prepared for the Department of the Environment, Heritage and Local Government (DEHLG). OSPAR Commission (accessed 19.02.16.). Hernández-Kantún, J.J., Riosmena-Rodriguez, R., Adey, W.H., Rindi, F., 2014. Analysis of the cox2-3 spacer region for population diversity and taxonomic implications in rhodolith- forming species (Rhodophyta: corallinales). Phytotaxa 190, 331–354. ˜ V., Maggs, Hernández-Kantún, J.J., Riosmena-Rodriguez, R., Hallspencer, J.M., Pena, C.A., Rindi, F., 2015. Phylogenetic analysis of rhodolith formation in the Corallinales (Rhodophyta). Eur. J. Phycol. 50, 46–61. Kaleb, S., Falace, A., Woelkerling, W.J., 2012. Phymatolithon lamii (Hapalidiaceae, corallinales, rhodophyta): a first report for the Mediterranean Sea. Bot. Mar. 55, 377–385. Konar, B., Riosmena-Rodriguez, R., Iken, K., 2006. Rhodolith bed: a newly discovered habitat in the North Pacific Ocean. Bot. Mar. 49, 355–359. Lemoine, M., 1910. Repartition et mode de vie du maërl (Lithothamnium calcareum) aux environs de Concarneau (Finistere). Monaco In: Annals De L’institut Oceanographique, 1, pp. 1–28 (3). McCoy, S.J., Kamenos, N.A., 2015. Coralline algae (Rhodophyta) in a changing world: integrating ecological, physiological, and geochemical responses to global change. J. Phycol. 51, 6–24. Mendoza, M.L., Cabioch, J., 1998. Étude comparée de la reproduction de phymatolithon calcareum (Pallas) adey & McKibbin et lithothamnion corallioides (P. & H. crouan) P. & H. crouan (Corallinales rhodophyta), et reconsidérations sur la définition des genres. Can. J. Bot. 76, 1433–1445. ˜ V., Hernández-Kantún, J., Le Gall, L., Bárbara, I., Barreiro, Pardo, C., Lopez, L., Pena, R., 2014. A multilocus species delimitation reveals a striking number of species of coralline algae forming maerl in the OSPAR maritime area. PLoS One 9, e104073, http://dx.doi.org/10.1371/journal.pone.0104073. ˜ V., Bárbara, I., 2004. Diferenciación morfológica y anatómica entre Pena, Lithothamnion corallioides y Phymatolithon calcareum (Corallinales, Rhodophyta) en dos bancos de maërl de la Ría de Arousa (N.O.Península Ibérica). Anim. Biol. 26, 21–27. ˜ V., Bárbara, I., 2008. Maërl community in the north-western Iberian Pena, Peninsula: a review of floristic studies and long-term changes. Aquatic Conserv. Mar. Freshw. Ecosyst. 18, 339–366. ˜ V., Bárbara, I., 2009. Distribution of the Galician maërl beds and their shape Pena, classes (Atlantic Iberian Peninsula): proposal of areas in future conservation actions of the Galician maërl. Cah. Biol. Mar. 50, 353–368. ˜ V., Hernandez-Kantun, J.J., Grall, J., Pardo, C., Lopez, L., Bárbara, I., Le Gall, L., Pena, Barreiro, R., 2014. Detection of gametophytes in the maërl-forming species Phymatolithon calcareum (Melobesioideae, Corallinales) assessed by DNA barcoding. Cryptogam. Algol. 35, 15–25. ˜ V., Pardo, C., López, L., Carro, B., Hernandez-Kantun, J., Adey, W.H., Bárbara, I., Pena, Barreiro, R., Le Gall, L., 2015. Phymatolithon lusitanicum sp. nov. (Hapalidiales: rhodophyta): the third most abundant maërl-forming species in the Atlantic Iberian Peninsula. Cryptogam. Algol. 36, 429–459. Pereira-Filho, G.H., Amado-Filho, G.M., Moura, R.L., Bastos, A.C., Guimarães, S.M.P.B., Salgado, L.T., Francini-Filho, R.B., Bahia, R.G., Abrantes, D.P., Guth, A.Z., Brasileiro, P.S., 2012. Extensive Rhodolith beds cover the summits of southwestern Atlantic ocean seamounts. J. Coastal. Res. 28, 261–269. Rosas-Alquicira, E.F., Riosmena-Rodrıguez, R., Couto, R.P., Neto, A.I., 2009. New additions to the Azorean algal flora, with ecological observations on rhodolith formations. Cah. Biol. Mar. 50, 143–151. Saitou, N., Nei, M., 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406–425. Shepherd, S., 2013. Ecology of Australian Temperate Reefs: The Unique South. Csiro Publishing. Stearn, W.T., 1973. Botanical Latin. History, Grammar, Syntax, Terminology and Vocabulary. Newton Abbott, David & Charles. Steller, D.L., Foster, M.S., 1995. Environmental factors influencing distribution and morphology of rhodoliths in Bahía Concepción B.C.S., Mexico. J. Exp. Mar. Biol. Ecol. 194, 201–212. Steneck, R.S., 1986. The ecology of coralline algal crusts: convergent patterns and adaptive strategies. Annu. Rev. Ecol. Syst. 17, 273–303. Suneson, S., 1958. Lithothamnion calcareum vid svenska västkusten. Botaniska Notiser 111, 197–199.

M.A. Wolf et al. / Aquatic Botany 134 (2016) 75–81 Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., Kumar, S., 2011. MEGA5 molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28, 2731–2739. Tamura, K., 1992. Estimation of the number of nucleotide substitutions when there are strong transition-transversion and G + C content biases. Mol. Biol. Evol. 9, 678–687. Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680. Villas-Bôas, A.B., Riosmena-Rodriguez, R., Tâmega, F.T.S., Amado-Filho, G.M., Maneveldt, G.W., Figueredo, M.A.O., 2015. Rhodolith-forming species of the subfamilies neogoniolithoideae and hydrolithoideae (Rhodophyta, corallinales) from Espírito Santo State, Brazil. Phytotaxa 222, 169–184.

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Wilks, K.M., Woelkerling, W.J., 1994. An account of southern Australian species of Phymatolithon (Corallinaceae, Rhodophyta) with comments on Leptophytum. Aust. Syst. Bot. 7, 183–223. Wolf, M.A., Maneveldt, G.W., Kaleb, S., Moro, I., Falace, A., 2015. Morphological and molecular characterization of Hydrolithon rupestre (Corallinaceae, Corallinales, Rhodophyta): first report from the Mediterranean Sea. Phytotaxa 224, 59–71. Woelkerling, W.J., Irvine, L.M., 1986. The typification and status of Phymatolithon (Corallinaceae, rhodophyta). Br. Phycol. J. 21, 55–80. Woelkerling, W.J., Irvine, L.M., Harvey, A.S., 1993. Growth-forms in non-geniculate coralline red algae (Corallinales, Rhodophyta). Aust. Syst. Bot. 6, 277–293. Woelkerling, W.J., 1988. The Coralline Red Algae: an Analysis of the Genera and Subfamilies of Non-geniculate Corallinaceae. British Museum (Natural History) & Oxford University Press London & Oxford.