European Journal of Phycology New insights into the cryptic diversity ...

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New insights into the cryptic diversity of the North Atlantic–Mediterranean ‘ Porphyra leucosticta ’ complex: P. olivii sp. nov. and P. rosengurttii (Bangiales, Rhodophyta) a

b

c

d

c

Juliet Brodie , Inka Bartsch , Chris Neefus , Sotiris Orfanidis , Troy Bray & Art C. Mathieson a

e

Department of Botany , Natural History Museum , Cromwell Road, London SW7 5BD, UK

b

Alfred-Wegener Institute for Polar and Marine Research , PO Box 120161, 27515 Bremerhaven, Germany c

Department of Plant Biology , University of New Hampshire , 46 College Rd, Durham, NH, 03824, USA d

National Agricultural Research Foundation , Fisheries Research Institute , 640 07 Nea Peramos, Kavala, Greece e

Department of Plant Biology and Jackson Estuarine Laboratory , University of New Hampshire , 46 College Rd, Durham, NH, 03824, USA Published online: 20 Feb 2007.

To cite this article: Juliet Brodie , Inka Bartsch , Chris Neefus , Sotiris Orfanidis , Troy Bray & Art C. Mathieson (2007) New insights into the cryptic diversity of the North Atlantic–Mediterranean ‘ Porphyra leucosticta ’ complex: P. olivii sp. nov. and P. rosengurttii (Bangiales, Rhodophyta), European Journal of Phycology, 42:1, 3-28, DOI: 10.1080/09670260601043946 To link to this article: http://dx.doi.org/10.1080/09670260601043946

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Eur. J. Phycol., (2007), 42(1): 3–28

New insights into the cryptic diversity of the North Atlantic–Mediterranean ‘Porphyra leucosticta’ complex: P. olivii sp. nov. and P. rosengurttii (Bangiales, Rhodophyta)

Downloaded by [Ingenta Content Distribution (Publishing Technology)] at 18:03 07 October 2014

JULIET BRODIE1, INKA BARTSCH2, CHRIS NEEFUS3, SOTIRIS ORFANIDIS4, TROY BRAY3 AND ART C. MATHIESON5 1

Department of Botany, Natural History Museum, Cromwell Road, London SW7 5BD, UK Alfred-Wegener Institute for Polar and Marine Research, PO Box 120161, 27515 Bremerhaven, Germany 3 Department of Plant Biology, University of New Hampshire, 46 College Rd, Durham, NH, 03824, USA 4 National Agricultural Research Foundation, Fisheries Research Institute, 640 07 Nea Peramos, Kavala, Greece 5 Department of Plant Biology and Jackson Estuarine Laboratory, University of New Hampshire, 46 College Rd, Durham, NH, 03824, USA 2

(Received 12 April 2006; accepted 30 August 2006) The existence of cryptic diversity in the ‘Porphyra leucosticta’ complex of the North Atlantic and Mediterranean was confirmed from a wide variety of recent and over 100-year-old herbarium specimens using plastid and nuclear molecular data in combination with karyological, morphological and ecological observations. The results revealed the presence of a new species, P. olivii sp. nov., within the P. leucosticta complex. Possible evidence for sexual reproduction was observed in a culture of P. olivii from the holotype site, Gulf of Thessaloniki in Greece, where n ¼ 2 chromosomes in male gametes and neutral spores, and 2n ¼ 4 in zygotospores. Archeospores and neutral spores also formed in culture providing evidence for possible asexual reproduction. The results also revealed the presence of P. rosengurttii, originally described from the north-western Atlantic, in both the north-eastern Atlantic and the Mediterranean, and enabled new morphological and ecological information to be obtained for this species. The rbcL sequences for P. olivii, including from an 1889 specimen of ‘Porphyra leucosticta’, differed by one transition between the north-western Atlantic and Mediterranean. The ITS1 of the holotype of P. olivii differed by a single bp transition from another specimen from the same location. The ITS1 of specimens from the northwestern Atlantic were identical but differed by 3-4 bp substitutions from the specimens from the Mediterranean. Partial rDNA SSU sequences were identical. In P. rosengurttii, rbcL sequences, including from specimens dated 1890 and 1906, differed by a single transition between both regions, the ITS1 had 92–100% similarity and partial rDNA SSU sequences were identical. Our study indicates that molecular, morphological and ecological evidence from a wide variety of samples (recent and old) and locations should be investigated before a new species is erected. The molecular comparison with old herbarium specimens also revealed that P. olivii and P. rosengurttii are not recent invaders to the Mediterranean but have been present for at least 100 years. Key words: chromosomes, cryptic diversity, ITS1, Mediterranean, molecular biology, morphology, North Atlantic, Porphyra leucosticta, rbcL, rDNA SSU

Introduction It is becoming increasingly apparent that there is considerably more species diversity in some groups of red algae than previously thought. With the range of approaches and tools now available, not least the ability to obtain molecular sequence data from both recently collected samples and historical collections, we are increasingly able to uncover cryptic diversity, resolve species concepts and taxonomic relationships, and gain a greater Correspondence to: Juliet Brodie. e-mail: [email protected]

understanding of species distributions in different geographical areas. This is well illustrated in Porphyra, which is one of the largest red algal genera with over 115 recognized species (Brodie & Zuccarello, 2007) and for which new species continue to be described (e.g. Brodie & Irvine, 1997; Lindstro¨m & Fredericq, 2003; Nelson et al., 2001; Neefus et al., 2002). Historically, identification of Porphyra species has been problematic because of the lack of morphological characters with which to differentiate taxa. Ecological information such as seasonality, life-cycle regulation and substratum

ISSN 0967-0262 print/ISSN 1469-4433 online/07/010003–28 ß 2007 British Phycological Society DOI: 10.1080/09670260601043946


J. Brodie et al. preference can be helpful, but it has often been insufficiently studied. The difficulty in understanding taxonomic relationships is compounded by how species concepts have been applied in different geographical regions. Porphyra leucosticta Thuret in Le Jolis in the North Atlantic and Mediterranean is a case in point. The species sensu lato is readily distinguished by the distinctive male gametangial sori, which appear as yellowish-white patches or streaks around the margins of the blades. It was originally described by Thuret in 1863 from Cherbourg, France (Le Jolis, 1863) and has since been recorded from the north-east and north-west Atlantic, from Canada to Uruguay, and from the Mediterranean and Black Sea (e.g. Zinova, 1967; Zavodnik & Zavodnik, 1973; Coll & de Oliveira Filho, 1976; Bird & McLachlan, 1992; Gargiulo et al., 1994; Flores-Moya et al., 1997), but it has become apparent that the name has been used for more than one species (Brodie & Irvine, 2003) in both the North Atlantic and Mediterranean. According to recent literature, there are ten species of Porphyra in the north-eastern Atlantic: P. amplissima (Kjellman) Setchell & Hus in Hus, P. birdiae Neefus & Mathieson, P. dioica Brodie & L. Irvine, P. drachii Feldmann, P. linearis Greville, P. leucosticta, P. purpurea (Roth) C. Agardh, P. miniata (C. Agardh) C. Agardh, P. umbilicalis (Linnaeus) Ku¨tzing and P. yezoensis sensu Kornmann (Brodie & Irvine, 2003; Brodie & Nielsen, 2005). This list comprises taxa for which there is type material and well-documented evidence on which to base a reliable species concept, although these authors noted that there were almost certainly unrecognized species in the region and that P. leucosticta probably represented a species aggregate. Prior to a critical examination of Porphyra taxa from New England and the Canadian Maritimes (Bray et al., 2006; Neefus et al., 2002; Klein et al., 2003; West et al., 2005), six species were recorded from the north-western Atlantic (Taylor, 1966; Hehre & Mathieson, 1970, 1993; Mathieson & Hehre, 1986; Bird & McLachlan, 1992; Sears, 2002): P. amplissima, P. linearis, P. leucosticta, P. miniata, P. purpurea, and P. umbilicalis. Most species were originally described from the northeastern Atlantic, but because of the geographical origin and difficulty of distinguishing morphologically between species, it is uncertain whether the names have been correctly applied to northwestern Atlantic material (Brodie et al., 1999; Klein et al., 2003). More recently, a seventh species, P. birdiae was added to the list (Neefus et al., 2002), and several other taxa were also delineated from Connecticut, within Long Island Sound (Neefus et al., 2000). Introduced

4 populations of the Asian species P. yezoensis were recently confirmed in New Hampshire (West et al., 2005). During studies of Porphyra (Rhodophyta, Bangiales) from the North Carolina coast of the United States, Coll & Cox (1977) noted that only two species were previously known, namely P. leucosticta and P. umbilicalis (Hoyt, 1920; Williams, 1948, 1949; Humm, 1979). On examining freshly collected material and herbarium specimens from North Carolina, however, they concluded that none of the material could be identified as these two species. Accordingly, they described two new taxa, P. carolinensis Coll & Cox and P. rosengurttii Coll & Cox and suggested that records of P. leucosticta and P. umbilicalis from other north-western Atlantic areas be re-examined. P. carolinensis was recently synonymized with P. suborbiculata Kjellman, along with P. lilliputiana Nelson, Knight & Hawkes (Broom et al., 2002). Accordingly P. suborbiculata is now regarded as a cosmopolitan, and possibly introduced, Asiatic species with disjunct populations in the Atlantic, Pacific and Indian Oceans (Masuda et al., 1991; Silva et al., 1996). Based on sequence comparison of the nuclear ribosomal small subunit gene (rDNA SSU) and the ribulose bisphosphate carboxylase (RuBisCO) large subunit gene (rbcL), one of the un-named Connecticut taxa was identified as P. suborbiculata (as P. carolinensis; Klein et al., 2003). A second Connecticut taxon, which was collected from the western part of the state at Cove Island Park, Stamford, was initially identified as P. leucosticta, but, based upon molecular data and an evaluation of North Carolina specimens, it now appears to be P. rosengurttii (Neefus et al., 2000, Blodgett et al., 2002). Porphyra species listed from the Mediterranean include P. atropurpurea (Olivi) de Toni, P. dioica, P. leucosticta, P. linearis, P. purpurea and P. umbilicalis (Furnari et al., 1999), although, as in the north-western Atlantic, there is uncertainty as to whether the species concepts of the north-eastern Atlantic have been correctly applied. P. atropurpurea (as Ulva atro-purpurea Olivi), which was originally collected from the Venice lagoon, was the earliest Porphyra species described from the Mediterranean (Olivi, 1794). De Toni (1897), who noted the similarity between P. leucosticta and U. atro-purpurea, proposed the combination P. atropurpurea and included P. leucosticta as a synonym. De Toni (1897) also included another species, P. coriacea Zanardini, which was also described from Venice (Zanardini, 1841), as a synonym of P. atropurpurea. Later, Feldmann (1939) noted that Olivi’s (1794) original description did not agree with that of P. leucosticta. Olivi wrote


Cryptic diversity in the Porphyra leucosticta complex that the blade had a small continuous whitish margin, which was interpreted by Feldmann as being similar to the male spermatangial sori of P. umbilicalis and thus synonymized P. atropurpurea with P. umbilicalis. Thus, the position of P. atropurpurea and also P. coriacea is still unclear. This is compounded because none of the three specimens illustrated in the original description (Olivi 1794, p. 154, tavs I–III) is designated as the type of P. atropurpurea. Herbarium material (Fig. 35) of the species in Olivi’s Herbarium in the Museo Storia Naturale in Venice superficially resembles Olivi’s (1794) tav II but appears to be composed of several individuals. Chromosome studies (Gargiulo et al., 1999) of male spermatangia in Porphyra material from the Venice lagoon revealed that n ¼ 6 (an unusual number for Porphyra species – see Brodie & Irvine 2003), and was interpreted as further evidence for a separate species of P. atropurpurea. A third Mediterranean species described from Croatia, P. cordata Meneghini, was described as small (blades 2–3 cm broad), cordate-reniform with distinct male gametangial sori (Meneghini, 1844) and epiphytic on Hypnea confervoides J. Agardh. The status of this species remains unknown. P. leucosticta was recorded by Thuret (Le Jolis, 1863) from both the Atlantic and the Mediterranean, although the type was collected from the former (Cherbourg in northern France). Hamel (1924) distinguished two forms of P. leucosticta, f. atlantica and f. mediterranea, in order to distinguish those from Cherbourg, which had irregular male gametangial patches typically in groups close to the edge of the frond, from smaller individuals in the Mediterranean which had striped male gametangial sori towards the centre of the frond. Feldmann (1939) further noted that Mediterranean material from Banyuls was mostly epilithic, forming a well developed community with other Porphyra species from winter to spring in the shallow sublittoral. This contrasted with the ecological conditions of north-eastern Atlantic material that grew mostly epiphytically in the intertidal and was confined to spring and summer. In 1986, material of a Porphyra, which had the characteristic male gametangial sori of P. leucosticta, was collected by Orfanidis from the Gulf of Thessaloniki in Greece. Another species collected near Malaga in southern Spain, which had for many years been investigated under the names of P. umbilicalis and P. leucosticta (e.g.: Hernandez et al., 1993; Figueroa, 1996; Figueroa et al., 1997; Flores-Moya et al., 1997), showed similar male gametangial sori. Electrophoresis, culture and chromosome studies indicated that these species were distinct from P. leucosticta and from each other (Bartsch pers. obs.; Orfanidis

5 2001). Molecular sequences of the RuBisCO spacer (rbcL-rbcS) and partial nuclear rDNA SSU showed that there were indeed two separate species and that they were distinct from P. leucosticta (Brodie & Bartsch, pers. obs.). The species from Spain was also later found in Greece. A comparison of the molecular data of specimens from the eastern USA (Neefus et al., pers. obs.) with those from the Mediterranean material indicated that one of the Mediterranean species appeared to match P. rosengurttii from North Carolina, while the other matched an undescribed taxon common along the coast of New England. In 2004, specimens with characteristic ‘leucosticta’ sori, collected from the south coast of England, were found to have molecular data that matched P. rosengurttii. In this paper we present the data that support these conclusions, describe a new species of Porphyra and provide new morphological, cytological, ecological and geographical information for P. rosengurttii from the North Atlantic and the Mediterranean. Materials and methods Collections Porphyra specimens were obtained from new field collections and old herbarium material. On the day of collection or as soon as possible thereafter, fresh specimens were floated out onto herbarium paper and in most cases a small tissue sample of each individual was removed and dried in silica gel for subsequent DNA analyses. Specimens from Spain and Greece were dried as whole individuals in silica gel and additional voucher specimens were pressed for morphological work. Field collections were made from: (i) North-western Atlantic (Fig. 1A): 100 sites from the western end of Long Island Sound, New York, USA to the north-eastern end of Nova Scotia, Canada; one site in Masonboro Sound, New Hanover County, North Carolina, USA near the type location for P. rosengurttii; two sites in the Gulf of Mexico: Port Aransas Jetty, Port Aransas, Texas, USA and San Jacinto Park, Galveston, Texas, USA. (ii) North-eastern Atlantic (Fig. 1B): Bracklesham Bay, Sussex, UK, Punta de Galdar, NW Canaries. (iii) Mediterranean Sea (Fig 1B): one site at Lagos, near Malaga, Spain; three sites in Greece: Palio and Cape Brasidas, Gulf of Kavala and N. Krini, Gulf of Thessaloniki. Historical collections were examined from: the Albion R. Hodgdon Herbarium, University of New Hampshire, Durham, New Hampshire (NHA); the Farlow Herbarium, Harvard University, Cambridge, Massachusetts (FH); Natural History Museum, London (BM); holotype and isotype specimens of P. rosengurttii: algal herbarium at the Smithsonian Institution, Washington, DC (US); isotype specimens of P. coriacea Zanardini: BM; photographs of the holotype and isotype, plus small tissue samples of the holotype: Museo Storia Naturale in Venice, Italy. Additional specimens were examined from: NY, PC, UPS, MICH


J. Brodie et al.

Fig. 1. Porphyra olivii sp. nov. and P. rosengurttii collection sites of investigated material. (A) NW-Atlantic: Maine, New Hampshire, Connecticut, Massachusetts, Masonboro Sound (North Carolina), Port Aransas and Isla Blanca (Texas) (all USA). (B) NE-Atlantic and Mediterranean: Canary Island, Malaga (Spain), Menorca (Spain), Trieste (Italy), Gulf of Thessaloniki (Greece), Bracklesham Bay (UK). Black triangles: P. olivii, black circles: P. rosengurttii. Scale bar: 400 km.

and L. Voucher specimens from new collections were deposited at NHA and BM. Herbarium abbreviations follow Holmgren et al. (1990).

Molecular methods Details of specimens used in molecular studies are shown in Table 1. Most of the molecular analyses were completed at the University of New Hampshire (UNH), with the exception of the rbcL–rbcS spacer of ten specimens and partial rDNA SSU sequence of eleven specimens, which were completed at the Alfred Wegener Institute for Polar and Marine Research in Bremerhaven, Germany (AWI).

Extraction and sequencing methods at UNH. Tissue samples (4 mm2) were ground in a mortar and pestle, and genomic DNA was extracted using a Puregene Genomic DNA Purification kit. For most samples, a 1,467-base-pair (bp) fragment, from position 67 (amino acid 23) of the large subunit of rbcL through the rbcL– rbcS intergenic spacer to the first codon of the small subunit, was amplified with rbcL primers F67 and rbc– spc (Teasdale et al., 2002). The rbc–spc primer is selective for the Bangiales and does not amplify

6 contaminating DNA from epiphytes that are common on macroalgal samples. PCR reactions were carried out as in Teasdale et al. (2002). For very old specimens (over 50 years old), the same region was amplified in smaller segments using primer pairs RBCL5–RC and rbc–spc, RBCL3 and RBCL5, RBCL5 and RBCL7, RBCL1 and RBCLSEQ, and F67 and RBCL6 (Klein et al., 2003, Teasdale et al., 2002). The rbcL–rbcS intergenic spacer of some specimens was amplified and sequenced using the primers Rub5 (50 -TGTGGACCTCTACAAACAGC-30 ; Maggs et al., 1992) and Rub3 (Brodie et al., 1996) or Rub3Por (50 -CCCATAATTCCCAGTA-30 designed by G. Barker (Robba et al., 2006). A portion of the rDNA SSU was amplified and sequenced using primers and PCR conditions described by Broom et al. (1999). The ITS1–5.8S–ITS2 region was amplified using the JBITS7 (Broom et al., 2002) and AB28 primers (Steane et al., 1991). For very old specimens, a shorter region was amplified using JBITS7 and ITS1-R primers. (50 -TATCCACCGTTAAGAGTTGTAT-30 ) In either case, ITS1 was sequenced using the JBITS7 and ITS1-R. The PCR reagents and amplification profile were identical to those used by Teasdale et al. (2002). The resulting amplicons were gel-purified to confirm their size and to decrease the presence of nonspecific or contaminating products prior to sequencing (Klein et al., 2003). PCR-amplified rbcL and ITS1 products were sequenced with an ABI 377 Automated Sequencer at the UNH Hubbard Center for Genome Sciences, using standard procedures as outlined in Klein et al. (2003). Raw sequence chromatograms were edited in Chromas (version 2.2, Technelysium, Pty. Ltd, Tewantin, Queensland, Australia). Contiguous sequence assembly was completed using SeqMan II (version 6.1 for Windows, DNAStar, Inc., Madison, Wisconsin, USA).

Extraction and sequencing methods at BM and AWI. DNA extraction and amplification methods followed those in Brodie et al. (1996, 1998). The RuBisCO spacer region was amplified using Rub5 and Rub3Por. Sequence data were obtained for the Xs region of rDNA SSU following the methods given in Broom et al. (1999) using primers GO4 (50 -CAGAGGTGAAATTCTTGGAT30 ), GO6 (50 -GTTGGTGGTGCATGGCCGTTC-30 ), JO4 (50 -AAACCTTGTTACGACTTCTCC-30 ) and JO5 (50 -ACAAAGGGCAGGGACGTATTC-30 ). Sequencing was undertaken by Qiagen, Germany. Sequence alignments and phylogenetic analyses. Alignments for all sequences were undertaken using the Clustal V or W methods in MegAlign (version 6.1 for Windows, DNAStar, Inc., Madison, Wisconsin, USA). Searches of GenBank were completed using BlastN via the Net Search option in MegAlign. The rbcL, rbcL–rbcS spacer and ITS1 sequences for representative specimens were submitted to GenBank. Phylogenetic analysis of trimmed rbcL sequences (1,188 bp, positions 215–1,402) from this study plus GenBank sequences for other Porphyra species from the North Atlantic was carried out via Bayesian and Neighbour Joining (NJ) Distance methods. Bayesian

43 320 39.4800 N; 70 190 56.100 W

Pine Pt. Landing, Scarborough, Maine, USA Dover Point, Newington, New Hampshire, USA Goose Cove, Gloucester, Massachusetts, USA

Yacht Club, Hull, Massachusetts, USA Humarock, Massachusetts, USA Bone Hill Rd. E. Barnstable, Massachusetts, USA New Bedford, Massachusetts, USA Judith Pt., Sand Hill State Park, Rhode Island, USA Hammonasset State Park, Connecticut, USA Seaside Park, Bridgeport, Connecticut, USA

45 390 N; 13 470 E 43 540 40.1200 N; 69 520 06.4200 W

Trieste, Adriatic, Italy New Meadows River, Brunswick, Maine, USA

0

00

0

00

0

00

Apr 2005 Apr 2005 Apr 2005 Jan 2005

41 140 57.2500 N; 72 320 43.0700 W 20 Feb 2005

41 080 34.0600 N; 73 130 41.9300 W

00

21 19 19 20

0

Mar 2004 Mar 2002 Mar 2002 Mar 2002 Mar 2002 Feb 2000 Feb 2002 Feb 2002 Mar 1889 Apr 2005 Apr 2005 Apr 2005 Apr 2005

10 Feb 2004 13 Feb 2004 3 Mar 2005 3 Mar 2005 3 Mar 2005 5 Apr 2005 5 Apr 2005 9 Apr 2005 3 Jan 2005

16 28 28 28 28 5 27 27 30 1 17 17 12

Date

41 36 53.04 N; 70 54 32.72 W 41 220 35.0200 N; 71 300 49.4100 W

42 080 14.0800 N; 70 410 38.4700 W 41 420 39.4700 N; 70 160 31.1400 W

42 180 25.6100 N; 70 530 27.2900 W

42 39 02.06 N; 70 40 20.09 W

43 070 10.4700 N; 70 490 38.4200 W

40 370 17.200 N; 22 560 50.100 E

40 370 17.400 N; 22 560 50.500 E

Porphyra olivii sp. nov. Taverna Chara, N. Krini, Thessaloniki Gulf, Greece

Hospital Panagia, N. Krini, Thessaloniki Gulf, Greece

Latitude & longitude

Taxon/location

Table 1. Collection information for specimens used in molecular evaluations.

Bray Bray Bray Bray & J. Day

A. Mathieson A. Mathieson A. Mathieson J. Day J. Day J. Day

T. Bray & D. Bray

T. T. T. T.

C. Neefus C. Neefus T. Bray & T. Bray & T. Bray & T. Bray & T. Bray & T. Bray & T. Bray

S. Orfanidis S. Orfanidis S. Orfanidis S. Orfanidis S. Orfanidis S. Orfanidis S. Orfanidis S. Orfanidis F. Hauck, P. Richter T. Bray & J. Day T. Bray & J. Day T. Bray & J. Day T. Bray

Collector

NHA77936

NHA78096 NHA78073 NHA78075 NHA77899

NHA78138 NHA78149 NHA77939 NHA77941 NHA77943 NHA78005 NHA78007 NHA78037 NHA77864

Holotype BM000806050 TES 22 TES 23 TES 25 TES 27 BM000806051 TES 12 TES 19 BM000806061 NHA77994 NHA78064 NHA78065 NHA78049

Specimen ID

DQ813612

DQ813624 DQ813622 DQ813623 DQ813611

DQ813625 DQ813626 DQ813613 DQ813614 DQ813615 DQ813617 DQ813618 DQ813619 DQ813610

DQ837009 DQ813616 DQ813621 DQ837010 DQ813620

DQ837008

DQ837007

rbcL

DQ834624

DQ8346221

DQ8346231

rbcL–rbsS spacer

GenBank/EMBL Accession Number


DQ813571

DQ813570

DQ813569

DQ813574

DQ813572

DQ813575

DQ834614

DQ834613

ITS-1

(continued)

DQ813587

DQ834637

DQ813588

DQ8346321

DQ8346331 DQ8346341 DQ8346351 DQ8346361

SSU

Cryptic diversity in the Porphyra leucosticta complex 7

41 010 18.5200 N; 73 310 16.8800 W

Shippam Pt. Stamford, Connecticut, USA

10 Mar 2005 10 Mar 2005 18 Apr 1906

27 500 N; 97 40 W

0

0

P. leucosticta00

0

0

36 480 N; 3 260 W

39 52 N; 04 14 E

50 450 N; 0 540 W

41 47 N; 71 22 W

00

34 100 N; 77 480 W

24 19 17 8 8 1

41 020 31.0800 N;73 300 04.7100 W

1

Sequenced by J. Brodie and I. Bartsch.

Apr 2000 Jan 2000 Feb 2002 Feb 2002 Feb 2002 Feb 2002 Feb 2002 Feb 2002 Feb 2002 1870 Jan 1980 2 Apr 2004 20 Mar 1999

13 3 17 17 17 17 17 17 17

2 May 2004 2 May 2004 1 May 1890

Feb 1999 Nov 1999 Nov 2003 Feb 2002 Feb 2002 Apr 1969

3 Apr 1976

34 430 N; 76 400 W

Adriatic (as ‘‘P. coriacea’’) La Favorita ad Ercolano, Italy (as ‘‘P. leucosticta’’) Cape Brasidas, Kavala Gulf, Greece 40 530 36.500 N; 24 200 8.300 E Punta de Galdar, 28 90 N; 15 390 W NW Gran Canaria

Masonboro Sound, North Carolina, USA Isla Blanca Park, Port Isabel, Texas, USA South Jetty, Port Aransas, Texas, USA Silver Spring, Providence, Rhode Island, USA Bracklesham Bay, Sussex, England Porto Mahan, Menorca, Baleares, Spain Lagos, Malaga, Spain

Porphyra rosengurttii Coll et Cox Pivers Island, Beaufort, North Carolina, USA Cove Is, Stamford, Connecticut, USA

30 30 16 16 16 16

41 020 31.0800 N; 73 300 04.7100 W

Cove Is, Stamford, Connecticut, USA Jan 2003 Jan 2003 Feb 2004 Feb 2004 Feb 2004 Feb 2004

Date

Latitude & longitude

Taxon/location

Table 1. Continued.

Neefus Neefus Yarish Yarish Yarish Yarish

J. Aguilera J. Aguilera J. Aguilera J. Aguilera J. Aguilera J. Aguilera J. Aguilera J. Aguilera J. Aguilera P. Titius G. Tripodi S. Orfanidis R. Haroun

I. Tittley I. Tittley J.J. Rodriguez

T. Bray T. Bray F.S Collins (5418)

C. Neefus C. Neefus C. Yarish, D. Snellgrove D.W. Freshwater, R. Melton D.W. Freshwater, R. Melton M.J. Wynne

J. Coll

C. C. C. C. C. C.

Collector

JB6 (specimen 1) JB4 (specimen 2) Mal 24 Mal 26 Mal 27 Mal 28 Mal 29 Mal 30 Mal 32 CDN1832 UMich JB2 (specimen no 3) CAN1

JB284 JB283 FH017

NHA77966 NHA77968 FSC 5418

NHA71368 NHA76230 NHA77093 NHA76229 NHA76234 Wynne 2426

US55293, Holotype

NHA78115 NHA78114 NHA78116 NHA78118 NHA78117 NHA78119

Specimen ID

DQ837020 DQ837021

DQ837019

DQ837018

DQ837017

DQ837016

DQ837015

DQ837014

DQ837013 AY486349

AF228754

DQ837012

DQ813631 DQ837011 DQ813627 DQ813628 DQ813629 DQ813630

rbcL

DQ834631

DQ834628 DQ8346291 DQ8346301

1

DQ8346271

DQ834626

DQ834625

rbcL–rbsS spacer

DQ834621 DQ8346431

DQ834620

DQ834619

DQ834618

DQ834615 DQ834617

ITS-1

GenBank/EMBL Accession Number


DQ8346411 DQ8346421

DQ8346381 DQ8346391 DQ8346401

SSU

J. Brodie et al. 8


Cryptic diversity in the Porphyra leucosticta complex Analysis was completed with MrBayes (version 3.1) using the General Time Reversible evolutionary model with the rate model varying according to codon position. It was run for 10,000,000 generations with a sampling frequency of 100 and burn-in set to 100,000 trees. The resulting tree was rooted using a GenBank sequence (AF087119) for Smithora naiadum (C.L. Anderson) G.J. Hollenberg as an outgroup. The tree was converted to Windows Meta-file (WMF) format using TreeView for Windows (version 1.6.6), and was prepared for final publication using Adobe Illustrator (version 9.0). A NJ Distance tree was constructed using PAUP (version 4 beta 10) with tie-breaking set to random. Reliability of the NJ tree was estimated by bootstrapping with 2,000 replicates. A consensus tree was computed using the 50% majority rule.

Morphological and ecological assessments In addition to the specimens listed in Table 1, the following specimens were examined for morphological work: P. olivii: Greece: Gulf of Thessaloniki, 5 March 2001, collected by (coll.) S. Orfanidis (two individuals); N. Krini, 5 February 2000, coll. S. Orfanidis (two individuals); P rosengurttii: Greece: Gulf of Kavala, Palio, 14 April 2004, coll. S. Orfanidis (two individuals); Spain: Lagos, near Malaga, December 1999, coll. J. Aguilera; Lagos, near Malaga, 1 March 2000, coll. J. Aguilera (seven individuals); Lagos, Malaga, 13 April 2000, coll. J. Aguilera (four individuals); Lagos, Malaga, 17 February 2002, J. Aguilera. Morphological features and ecology were compared (Tables 2, 3; cf. Neefus et al., 2002). Colour was recorded at several positions on each blade, using an X-Rite Digital Swatchbook Colorimeter; the values were averaged in Colorshop v.2.6.0 (X-Rite, Grandville, Michigan, USA). Colour is expressed in CIE L*a*b* tristimulus units, which are based on a ‘standard observer’ and are device independent (Bunting, 1998). Herbarium sheets used for figures were scanned (Epson 1640XL or Agfa Snapscan E40) and edited without changing colour appearance (Adobe Photoshop CS). Seasonal, geographic and vertical distributions were enumerated from herbarium label data, field notes, published information and observation of in situ populations. Blade thickness, chloroplast morphology, number of cell layers, division sequences of male gametangia and zygotosporangia and number of chromosomes were assessed from microscopic examination of surface view (SV) and transverse sections (TS) of blades, semi-thin sections or squash-preparations (UNH: Olympus BX40 microscope, digital camera: Nikon D100; AWI: Zeiss Axiophot photomicroscope, Agfa APX25 film for black and white prints, scan of negatives: CanonScan FS 2710 scanner; Fisheries Research Institute, Kavala, Greece (NAGREF-FRI): Olympus BH 2-PM20 photomicroscope, Agfa APX25 film for black and white prints). The terminology used for reproductive structures follows Nelson et al. (1999). Reproductive parts of fresh specimens of P. rosengurttii from Spain (collected Dec. 1999) and of cultured material of P. olivii from Greece

9 were investigated using semi-thin sections. These were established by following the protocol of Wiencke & Clayton (1998).

Karyology P. olivii, culture material (culture No. 2507; see below) grown at 20 C (0.5 C), long-day (LD; 14:10 h light: dark cycle) and short-day (SD; 10:14 h light:dark cycle) conditions at 20 mmol m2s1 was fixed in 3:1 ethanol– glacial acetic acid at 4 C, left overnight and stained with Schiff’s reagent following the protocol of Pijnacker & Ferwerda (1995). Wild material of P. rosengurttii from Spain (collected Feb., March and Dec. 1999) was transported to Bremerhaven, Germany, in a cooled styropor box, and fixed in fresh 3:1 ethanol–glacial acetic acid. The material was stained with Wittmann’s aceto-iron haematoxylin-chloral-hydrate technique (Wittmann, 1965) using the protocol of Holmes & Brodie (2005).

Culture material A clonal culture of P. olivii was established in 1986 by S.O. (AWI Culture No 2507; collection Lu¨ning) from asexual spores under the name of P. leucosticta (Orfanidis, 1991; Orfanidis & Haritonidis, 1996; Orfanidis, 2001). The blade phase of this culture was grown in aerated 2-l glass beakers in von Stosch enriched seawater (VSES, modified according to Guiry & Cunningham, 1984) in 10 and 15 C, LD (16:8 h light:dark cycle) and SD (8:16 h light:dark cycle) conditions at 20 mmol m2s1. Fertile specimens were investigated for asexual reproduction and fixed for semithin sectioning (see above).

Results P. olivii sp. nov. Orfanidis, Neefus et Bray DIAGNOSIS Lamina monostromatica, tenuis, 15–30 m crassa, minute stipitata, basibus leviter vel profunde cordata, interdum pseudoumbilicata, 2–18 cm longa, 1–12.5 cm lata, orbiculata vel suborbiculata vel late-ovata vel lanceolata, non laciniata, margines integra vel irregularis, levitus ad modice plicatus praecipuae in proximale parte e lamina, cellulis vegetatii a viso superficali 8–18 4–10 m. Laminae semi-translucentiae, coloris vegetatii variabilis: brunnescens, aereus, rubiginosus vel leviter olivaceous vel viridibrunnescens vel violaceous. Thalli monoecii, gametangiis feminis et masculinis in soris discretis separatis sed contiguis, marginalibus, distaliter latescentibus. Sori masculini luteoli-albi, aut in liniis continuus aut interruptis, rhombiformis vel irregularis vel striatis, 2–11 (15) mm longis, ad 4 mm latis, a viso superficali gametangiis masculiniis in massis dispositis 9–17 m latis et 12–25 m


J. Brodie et al. longis, in 4 stratis a 8 spermatis, in toto quoque 32 spermatis instructis. Sori femineis non conspicui, massis 10–27 m latis et 18–33 m longis a viso superficali, in 2 stratis a 4 zygotosporiis, in toto quoque 8 zygotosporiis instructis. Numero chromosomalis n ¼ 2. Thalli epiphytici in varius algae, zona sublittoralae vodosi habitans, annualis, in hieme et vere crescents. Numero Genbankii Holotypici: DQ837007, DQ834613. Gametangial blade monostromatic, thin, 15–30 mm in TS, minutely stipitate, bases slightly to deeply cordate, sometimes pseudoumbilicate, 2– 18 cm long by 1–12.5 cm wide, orbicular, suborbicular to broad-ovate or oval to lanceolate, small specimens obovate, not laciniate, margins entire to irregular; slightly to moderately ruffled mainly in lower part of blade, vegetative cells in SV 8–18 4–10 mm. Blades semi-translucent, vegetative colour variable: light brown, bronze, brownish-red to slightly olive-green or greenish-beige or violet. Monoecious, reproductive area continuous, marginal, from base to top, broadening distally; male gametangial sori yellowish-white either continuous or patchy, diamond to irregular shaped or in streaks, 2–11 mm (up to 15 mm) long and up to 4 mm broad, male gametangial packets in 4 tiers of 8 (32 spermatia), 9–17 mm wide 12–25 mm long in SV. Zygotosporangial sori initially inconspicuous, becoming reddish, in between but adjacent to the male sori, packets in 2 tiers of 4 (8 zygotospores), 10–27 mm wide 18–33 mm long in SV. Chromosomes n ¼ 2. Blades epiphytic on various algae, sublittoral fringe, seasonal annual, winter to spring. Genbank numbers of Holotype: DQ837007, DQ834613. HOLOTYPE: Collected as an epiphyte of Gracilaria sp. from Taverna Chara, N. Krini, Thessaloniki, Greece, by S. Orfanidis, 16 March 2004, shallow subtidal. Monoecious thallus (Fig. 2) dried on a single herbarium sheet; deposited in the Natural History Museum, London, UK (BM). ISOTYPES: Fisheries Research Institute, Kavala (Greece), University of Copenhagen, Copenhagen (C), Smithsonian Institution, Washington DC (US), University of New Hampshire, Durham (NHA), Natural History Museum, London (BM), Museum of New Zealand, Wellington (WELT), Hokkaido University, Sapporo (SAP), University of British Columbia, Vancouver (UBC), University of Messina (MS), University of Malaga (MGC). ETYMOLOGY: The name olivii is after G. Olivi (1769–1795), who was an Italian naturalist and the first to describe a Mediterranean Porphyra species, namely ‘Ulva atro-purpurea’ (Olivi, 1794). DESCRIPTION: The gametangial thallus is foliose (Figs 2–5) with a minute discoid holdfast and stipe that expands into a single, very thin translucent

10 blade, that is orbicular, sub-orbicular, broad ovate to lanceolate (small individuals, 20 mm in length, typically ovate) in shape, with a slightly to deeply cordate base, the lobes may overlap in larger specimens to give an umbilicate appearance. The blade, which is not laciniate, is up to 180 mm long, up to 125 mm broad and 15–30 mm thick, light brown, bronze, brownish-red, slightly olive-green, greenish-beige or violet in colour. The blade is slightly to moderately ruffled, mainly in its lower part. The blades are monostromatic and parenchymatous in structure (Figs 6, 7). In the central part, vegetative cells are 8–20 mm 4–16 mm in SV (Fig. 6) and 6–22 mm tall in TS (Fig. 7). The species is monoecious; male gametangia and zygotosporangia occur in separate soral patches but intermixed and adjacent to each other (Fig. 8), typically concentrated around the blade margin (Figs 3, 5). Male gametangia form a continuous band, c. 1 mm broad, from the cordate base, expanding distally to give a more flecked appearance (Fig. 2). As the blade expands, the male margin becomes discontinuous making the sori appear submarginal. Male gametangial sori are yellowish-white, initially diamond-shaped, becoming irregular, 2–10 mm long and up to 5 mm broad (Figs 2, 3). Where the male margins have disintegrated the blade becomes more irregular in shape. Zygotosporangial sori are inconspicuous, distinguished as irregularly shaped reddish patches, slightly darker than the vegetative blade and adjacent to male gametangial sori. In SV, male gametangia (Fig. 8) occur in packets of 4 (2 2), 9–17 mm wide 12–25 mm long, each dividing periclinally to give 8 cells in TS (Fig. 9), giving 32 male gametes in each packet. Zygotosporangia (Figs 8, 10) occur in packets of 4 (2 2), 10–27 mm wide 18–33 mm long in SV and 18–28 mm high in TS (Fig. 11), each dividing periclinally to give 2 cells in TS, giving 8 zygotospores in each packet (Fig. 11). Asexual sporangia were not observed in field-collected material. The conchocelis phase was not recorded in the field. On release spores were amoeboid and consisted of two different size classes, germinating either bipolarly into blades or unipolarly into conchocelis. The blade phase is epiphytic on other algae, notably on Gracilaria longa Gargiulo, De Masi & Tripodi, but also on Chondrus crispus Stackhouse, Dumontia contorta (S. G. Gmelin) Ruprecht and Fucus vesiculosus Linnaeus, although on one occasion it was recorded from rope. It is typically shallow subtidal at the type locality, but has been recorded from the midlittoral in the USA. Present geographical records (Fig. 1) and ecophysiological data of the

11


Cryptic diversity in the Porphyra leucosticta complex

Figs 2–5. Porphyra olivii sp. nov. Fig. 2. Holotype, collected Taverna Chara, N. Krini, Gulf of Thessaloniki, 16 March 2004 by S. Orfanidis, specimen No. 8, on Gracilaria sp. Fig. 3. Fresh material of same collection as Fig. 2. Fig. 4. Non-reproductive material, possibly young, collected N. Krini, Gulf of Thessaloniki, 8 February1990 by S. Orfanidis on Gracilaria sp. Fig. 5. NW-Atlantic reproductive material, collected Judith Pt., Sand Hill State Park, Rhode Island, USA. 19 April 2005 by T. Bray on Chondrus crispus, shallow subtidal. Scale bars: 2 cm (Figs 2–4) and 5 cm (Fig. 5).


J. Brodie et al.

12

Figs 6–11. Porphyra olivii sp. nov. Vegetative and reproductive anatomy of fresh blades. Fig. 6. Vegetative cells, surface view (SV). Fig. 7. Vegetative cells, transverse section (TS). Fig. 8. Mixed sorus of male and female gametangia, SV. Fig. 9. Male gametangia, TS. Fig. 10. Mature, female zygotosporangia with releasing zygotospores at blade margin, SV. Fig. 11. Female zygotosporangia, TS. Scale bars: 30 mm (Figs 6, 8, 10) and 25 mm (Figs 7, 9, 11).

Thessaloniki strain (Orfanidis, 2001) indicate a temperate amphi-Atlantic to Mediterranean distribution. The blade phase is a seasonal annual, occurring from December to mid-April in the Mediterranean (Haritonidis, 1978; October 1989 and October 1990; Orfanidis pers. obs.), and January–April in the USA. It appears to be present when the surface water temperature is below 20 C and the daylength is 514 h (Orfanidis, 2001). For USA material, all reproductive samples were in April. Herbarium specimens after drying become slightly glossy, pale brownish-pink, rose pink to pink with a hint of blue (CIE units-L: 69.18, a: 7.25, b: 33.32), the colour deepening to the margins, especially if fertile. Specimens adhere

very well to paper. The texture of herbarium specimens is smooth. In culture, blades that developed from culture no. 2507 and were subjected to 10 C and 15 C SD and LD conditions developed archeospores (18–24 mm in diameter) in 10 C LD intermixed with male gametangial packets (Fig. 12), and neutral spores (5.3 6.8 mm in SV, 14 mm high in TS) in packages of eight in SV at 15 C SD (Figs 13, 14); both spore types germinated into blades. The chromosome number in male gametes and neutral spores was n ¼ 2 (Figs 15, 17), and in zygotospores was 2n ¼ 4 (Fig. 16). Morphological and ecological features of the holotype from Greece and specimens from the USA plus P. leucosticta from Great Britain are compared in Table 2.



13

Figs 12–14. Porphyra olivii sp. nov. Asexual reproduction in culture. Fig. 12. Thallus margin with archeospores adjacent to male gametangial packets in surface view (SV) at 10 C, long-day conditions. Fig. 13. Thallus margin with neutral sporangia in packages of eight spores (arrow) in SV at 15 C, short-day conditions. Fig. 14. Neutral spores in transverse section. Scale bars: 50 mm (Figs 12, 13) and 25 mm (Fig. 14).

Comparison of P. rosengurttii in North-Atlantic and Mediterranean populations Comparative data of the type and other specimens of P. rosengurttii from North Carolina and of the species from elsewhere in the North Atlantic (Connecticut, USA and Sussex, UK) and Mediterranean (Lagos, Spain and Kavala, Greece) are shown in Table 3. The shape of the thallus can be orbicular/sub-orbicular to sublanceolate (Figs 18–25); only the western Atlantic material is reniform. Material from all localities is cordate to pseudoumbilicate. Specimens that frequently show slightly off-centre bases from the longitudinal axis (Fig. 20) are found at all localities. Western North Atlantic material is not laciniate, whereas eastern North Atlantic material

Figs 15–17. Porphyra olivii sp. nov. Chromosomes from cultured material. Fig. 15. Male spermatia n ¼ 2. Fig. 16. Zygotospore 2n ¼ 4. Fig. 17. Neutral spores n ¼ 2. Scale bars: 10 mm (Figs 12–14).

and Mediterranean specimens are occasionally laciniate. Sussex specimens collected in May and those from Spain collected late in the season are mostly small, pseudoumbilicate, and necrotic at the edges of the blade (Figs 23, 24). Margins of specimens from all localities are always undulate

2–14 1.5–12.5 cm Average: 1.5 Range: 0.8–3 (N ¼ 31) Entire; becoming irregular when male sori disintegrate; slightly to moderately ruffled, more so in lower part of blade Slightly to deeply cordate; sometimes pseudoumbilicate Monoecious; spermatangia near the base form a narrow, continuous band, becoming increasingly patchy or streaky towards the distal end, there extending sub-marginally; sori diamond to irregular-shaped or streaks, 2–11 mm (up to 15) long and up to 4 mm broad; zygotosporangial sori distinctly more reddish than the vegetative thallus forming a marginal zone between the male sori that is usually broader at the distal end Very good

15–30 mm Monostromatic SV: 7.5–20 4–15; TS: 6.25–11.25 mm tall

Dimensions (L B) L:W Ratio

Adherence to paper on drying

Cell Morphology Vegetative: Thallus thickness Cell layers Cell dimensions (L W mm)

Fertile area

Base

Margins

Orbicular to broad-ovate or oval, not laciniate

Fresh material: brownish red with a touch of olive green or brownish red to violet. Dried material: Pink with a hint of blue or greyish with a hint of purple

Shape

General morphology Colour

P. olivii Thessaloniki (Type)a

16–28.5 mm Monostromatic SV: 9.8–17.2 7.5–16; TS: 11–22 mm tall

Very good

Minutely stipitate; sometimes very slightly cordate Monoecious; spermatangia spots or streaks; mainly distal; zygotosporangial sori inconspicuous, marginal to sub-marginal

2.5–18 1–7 cm Average: 2.4 Range: 1.2–7.5 (N ¼ 26) Entire, slightly to moderately ruffled; with occasional tears

Fresh material: light tan (brown) to greenish beige (L: 69.18, a: 7.25, b: 33.32) in vegetative area; bronze to light green (L: 70.56, a: 2.39, b: 27.37) near the holdfast. Dried material: Pale brownish pink, rose pink to pink with a hint of blue Ovate, obovate to lanceolate, not laciniate

P. olivii New England, USAb

P. leucosticta Great Britainc,

d

34–50 mm Monostromatic SV: 8–24 6–10; TS: 36–38 mm tall SV: 10–16 8–12; TS: 16–32 mm talle

Very good

Minute discoid holdfast and stipe; often with a cordate base Monoecious; spermatangia irregular, lozenge-, slit- or occasionally diamond-shaped patches, up to 30 mm in extent; zygotosporangial sori inconspicuous, often adjacent to male sori

Entire; usually with some ruffles extending from centre

Elliptical, ovate, oblong, orbicular, linear or irregular, occasionally falcate, elongate or umbilicate, not usually laciniate Up to 22 (42) 20.2 (22) cm ND

Fresh material: chocolate brown, purple-mauve to light brown. Dried material: purple-mauve, smooth and glossy

Table 2. Comparison of Porphyra olivii sp. nov. from two locations in the Mediterranean and NW-Atlantic with P. leucosticta from Great Britain.


J. Brodie et al. 14

Epiphytic on coarsely branched species, mainly on Gracilaria longa Gargiulo, De Masi & Tripodi; forming an epiphytic community with other species (Ulva sp., Punctaria latifolia Greville, Polysiphonia sp.) on boulders surrounded by sand Sheltered

Patchy to dense, may cover host entirely during main growth period

Substrata

Abundance

Semi-exposed to exposed; one site estuarine tidal rapid Not abundant; scattered individuals and occasional clusters

Occasionally mid littoral; typically shallow subtidal Epiphytic on Fucus vesiculosus, Gracilaria tikvahiae McLachlan, Chondrus crispus and Dumontia contorta; one specimen on rope

Seasonal annual: Jan. to April; all reproductive samples were in April

ND

ND

Can appear as masses on Fucus serratus on some shores; otherwise scattered

Semi-exposed

Epiphytic and epizoic on mussels, Fucus serratus Linnaeus, Chondrus crispus and Mastocarpus stellatus (Stackhouse) Guiry, occupying different shore levels over the season; mid to lower intertidal

Seasonal annual: mainly spring to summer: (March) April to Sep. (Oct. to Dec.); reproductive throughout season Mid to lower littoral, shallow subtidal

Male gametes n ¼ 3–4, sometimes in the same blade

Not observed in field material; occasional archeospores or neutral spores in culturee,g

ND 2 tiers of 4; (8 zygotospores) SV: 16–20 21–30; TS: ND SV: 14–24 20–32e 10–18 mm in diameter on release

ND

ND 8 tiers of 8; (64 male gametes) SV: 12–14 16–22; TS: ND SV: 10–14 16–24e

a Measurements from dried material; bmeasurements from fresh material; cmeasurements combination of fresh and dried material; ddata from Brodie & Irvine (2003); eDevon, UK: data from Holmes & Brodie (2005); fOrfanidis (2001); gKornmann (1961). Abbreviations: B: breadth; L: length; L*a*b*: CIE tristimulus units for colour measurements, for further detail see Material and methods; n: haploid chromosome number; N: number of samples measured; ND: no data; SV: surface view; TS: transsection; n: haploid chromosome number.

Exposure

Shallow subtidal (15–20 cm depth)

Seasonal annual: Dec.–mid to April; from mid March to April maximal growth

Male gametes and archeospores n ¼ 2; zygotospores 2n ¼ 4

Not observed in field material; archeospores and neutral spores in culturef, this study

ND

SV: 4.9 9.8 mm; TS: 12.3 mm tall

23.4 mm (N ¼ 1) 2 tiers of 4; (8 zygotospores) SV: 9.7 19.7; TS: 24.7 mm tall

25–35 mm 2 tiers of 4; (8 zygotospores) SV: 15–27 18–33; TS: 17.5–27.5 mm tall

ND

22.2–28.4 mm 4 tiers of 8 (16); (32 male gametes) SV: 11.1–17.2 17.2–24.7; TS: 13.5 –18.5 mm tall 2.4–4.9 mm in diameter

21.3–32.5 mm 4 tiers of 8; (32 male gametes) SV: 9–15 12–21; TS: ND

Shore level

Ecology Seasonality

Cytology Chromosome number

Asexual reproduction Observations

Zygotospore dimensions

Gamete dimensions Mature zygotosporangia: Thallus thickness Packet arrangement Packet dimensions (L W mm)

Mature male gametangia: Thallus thickness Packet arrangement Packet dimensions (L W mm)



16


J. Brodie et al.

Figs 18–25. Porphyra rosengurttii. Morphological variation of habit at different localities in the North Atlantic and Mediterranean Sea. Fig. 18. Holotype of Coll & Cox (1977) from Beaufort, North Carolina, USA. Fig. 19. Two specimens collected 8 February 2002 at Masonboro Sound, North Carolina, USA by D.W. Freshwater and R. Melton. Fig. 20. Colour of fresh (above) and dried (below) specimens collected 19 November 1999 from Cove Island, Connecticut, USA by C. Neefus. Arrowhead points to off-centre holdfast. Fig. 21. Obovate specimen with typical long streaks of male gametangial sori (arrows) from Palio, Gulf of Kavala, Greece, collected 14 April 2004 by S. Orfanidis. Fig. 22. Laciniate form from Palio, Gulf of Kavala, Greece collected 14 April 2004 by S. Orfanidis. Fig. 23. Specimen from Lagos, Spain, with pseudoumbilicate habit and tear, collected 13 April 2000 by J. Aguilera. Fig. 24. Two specimens from Bracklesham Bay, Sussex, UK, collected 2 May 2004 by I. Tittley. Fig. 25. Specimen from Gran Canaria collected 4 February 1999 by R. Haroun. Scale bars: 2 cm (Figs 18, 20–25) and 5 cm (Fig. 19).

Undulate and folded, entire or laciniate Sessile, one to few blades attached by a central holdfast

Monoecious; spermatangia in irregular patches near margin; not mixed with other kinds of cells

Good

Margins

Fertile area

Adherence to paper on drying Cell Morphology Vegetative: Thallus thickness Cell layers Cell dimensions (L W mm)

Mature male gametangia: Thallus thickness Packet arrangement Packet dimensions (L W mm) Gamete dimensions Mature zygotosporangia: Thallus thickness Packet arrangement 32–37.5 mm 4 tiers of 4 (or 8) SV: 7.5 12.5 3–5 mm in diameter 32–37.5 mm 2 tiers of 2 (or 4) (4–8 zygotospores)

ND 1 (or 2) tiers of 4 cells (4–8 zygotospores)

32–37.5 mm Monostromatic V: 5–10 10–17.5 TS: 17.5–22.5 mm tall

5–14 3.5–10 cm Average 1.4 Range 1.0–2.6 (N ¼ 28) Ruffled, entire, lobed or laciniate Cordate to pseudoumbilicate; attachment point frequently off-centre relative to plant axes Monoecious; spermatangia in geometrically shaped patches at thallus margin; zygotosporangia in small clusters interspersed with vegetative cells near spermatangial sori Good, except when very young

Round, ovate to reniform

Olive-brown, becoming purple, red or pale mauve with age on drying

Connecticut, USAc

ND 2 (or 4) tiers of 16 ND 5 mm maximally

45–55 mm Monostromatic SV: 9 13 TS: 9–18 mm tall

16 8 cm ND

Dimensions (L B) L:W Ratio

Base

Reniform, oblong, sublanceolate, or sometimes cordiform

Brownish-red, olivaceous to violaceous

b

Shape

General morphology Colour

North Carolina, USAa,

40–52.5 mm 4 tiers of 4 (16 zygotospores)

40–50 mm 8 tiers of 8 SV: 20–30 15–25 ND

34–38/47.5–62.5 mm Monostromatic SV: 8–20 5–15 TS: 15–25 mm tall

Monoecious; spermatangia narrow streaks elongating in direction of blade expansion, occasionally block-like appearance, surrounded by marginal zygotosporangial zone Moderately good

Dried specimens: greyish to light olive-green; margins becoming brown-reddish in fertile specimens Orbicular, to broad-ovate to elongate, occasionally oblong to laciniate or lobed 1.5–14 1.5–10 cm Average 2.2 Range 0.8–7.7 (N ¼ 54) Moderately ruffled, entire to laciniate Cordate to pseudoumbilicate

Lagos, Spainb

Sussex, UKb

32.5–40 mm 2 tiers of 4 (8 zygotospores)

37.5 mm 8 tiers of 8 SV: 17.5–25 12.5–15 ND

25–35 mm Monostromatic SV: 7.5–20 7.5–12.5 TS: 12.5–25 mm tall

Moderately good

Monoecious; spermatangia narrow to broad patches, sometimes block-like appearance; zygotosporangial sori inconspicuous

2–11.5 1.8–4.5 cm Average 1.3 Range 0.67–3.8 (N ¼ 16) Entire or rolled; moderately to deeply ruffled Cordate to pseudoumbilicate

Suborbicular to elongate, occasionally laciniate

Fresh specimens: light brown; dried specimens greyishmauve

Table 3. Porphyra rosengurttii. Comparative morphology and ecology from locations in the N. Atlantic and Mediterranean Sea.


31 mm 2 tiers of 4 (8 zygotospores) (continued)

33–53 mm (mean: 37mm) 8 tiers of 8 (or 16) SV: 15–25 18–33 ND

27–42 mm Monostromatic SV: 15–32 4–9 TS: ND

Good

7.5–12 3.5–10.5 cm Average 1.6 Range 1.1–2.8 (N ¼ 12) Undulate, moderately ruffled, entire to laciniate Cordate to pseudoumbilicate; individual blades or as clusters of blades arising from single point Monoecious; spermatangia narrow streaks elongating in direction of blade expansion, surrounded by marginal zygotosporangial zone

Dried specimens: greyish with a hint of purple; becoming more purple towards ferlile margins Suborbicular to elongate to lanceolate, partly laciniate

Kavala Gulf, Greeceb


Sheltered coastal ND

Exposure Abundance

a

Sheltered coastal Conspicuous and fairly abundant as individual plants

On rocks in upper littoral, epiphytic on Chondrus crispus and Fucus vesiculosus in the mid- to lower littoral

Seasonal annual: Nov. to Feb., uncertain records from April and July Upper to lower littoral

ND

Not observed in field material

SV: 10 17 TS: ND SV: 8–12 11–15 mm TS: 8–12 mm tall

Connecticut, USAc

Exposed Conspicuous; in Feb. dense coverd

On slate rocks

Seasonal annual: Dec. to March (April to May); dominance in Jan. to March Upper littoral

Male gametes: mostly n ¼ 4, sometimes 3 or 2

Not observed in field material but only in culture

SV: 22.5–35 15–27.5 TS: ND ND

Lagos, Spainb

Semi-exposed Individuals scattered

On wooden sea defences (groynes) and on barnacles on defences

Upper to mid littoral

Unknown: May (one collection only)

ND

ND

SV: 17.5–25 15–22.5 TS: 20–22.5 mm tall ND

Sussex, UKb

Type material; data from Coll & Cox (1977); bmeasurements from dried material; cmeasurements from fresh material; dsee Hernandez et al. (1993). Abbreviations: see Table 2.

Substrata

Upper intertidal fringe or lower (intertidal?) On rocks and Crassostrea virginica (Gmelin) in upper littoral or lower on Gracilaria tikvahiae, and on bridge pilings

Shore level

Seasonal annual: Jan. to April

Ecology Seasonality

b

ND

‘Monospores’ at margin of thallus

9–11 18–20 mm

ND

North Carolina, USAa,

Cytology Chromosome number

Asexual reproduction Observations

Packet dimensions (L W mm) Zygotospore dimensions

Table 3. Continued.


Seasonal annual: Jan. to May; dominance from end of Feb. to mid-April Mid littoral to shallow sublittoral On rocks and boulders in midlittoral; in sublittoral fringe zone also epiphytic on Corallina elongata Ellis & Solander and Cystoseira crinitophylla Ercegovic Moderately exposed Conspicuous; in Feb. and March dense cover

ND

ND

SV: 15–28 18–35 TS: ND ND

Kavala Gulf, Greeceb

J. Brodie et al. 18


Cryptic diversity in the Porphyra leucosticta complex and moderately to deeply ruffled and, except in Sussex material where they are entire or rolled, entire, lobed or laciniate. Fresh and dried material from Lagos (Malaga, Spain) may also show inrolled margins. Most specimens are slightly longer than broad. Thallus thickness is variable, although the type material is thicker than specimens from all other localities. Colour is variable throughout the distribution area and season, changing with time after drying (Fig. 20) but almost always with a pinkish tone (Figs 18–25); material from Sussex (UK) and Greece is more greyish-mauve after drying than western Atlantic specimens. Male gametangial sori differ in shape between localities: North Atlantic specimens have irregular to block-like patches (Fig. 24), whereas Mediterranean material has conspicuous long, radially orientated streaks, giving a striated appearance (Figs 21, 22). Male gametangial sori are either as thick as (Connecticut) or thicker (UK and Mediterranean) than the vegetative thallus. The zygotosporangial sori thickness is similar to that of the vegetative thallus. The arrangement of the male gametangial packets varies between localities, giving between 32 and 128 (Figs 26, 27, 31). Developing zygotoporangia are shown in Figs 28–30. The arrangement of zygotosporangial packets (Figs 28–30) also varies between locations with 4–8 zygotospores in western North Atlantic material (Fig. 30), 8 in the eastern North Atlantic, and 8 to 16 in the Mediterranean (Figs 32, 33, Table 3). Porphyra rosengurttii is a seasonal annual, occurring from winter to spring in sheltered to semi-exposed conditions in the upper to mid-littoral and is usually epilithic, although epiphytic specimens are also present in the western North Atlantic in the lower littoral. Freshly collected specimens from Lagos (Malaga, Spain) were put into culture and followed on two occasions. On the first occasion, individual thalli, collected on 1 March 2000, released two size classes of spores. Individual spores either developed into the conchocelis or blade phase, with the larger of the two types of spores tending to produce blades. On the second occasion, individuals collected on 13 April 2000 released only spores of the smaller size class, which germinated into the conchocelis phase. The chromosome number is only known for material from Lagos (Malaga, Spain) which has n ¼ 4 in male spermatia (Fig. 34). Molecular analyses Partial rbcL, rbcL–rbcS spacer, ITS1 and/or partial rDNA SSU sequences were obtained from P. olivii and P. rosengurttii specimens from 30 locations in

19 the North Atlantic, Gulf of Mexico and the Mediterranean (Table 1). Porphyra olivii. Almost the entire rbcL gene (positions 73–1467) plus the rbcL–rbcS spacer (77 bp) was sequenced from the holotype specimen (BM000806050). The same region was sequenced for a second specimen (BM000806051) from the type location in the Gulf of Thessaloniki, plus three specimens from the north-western Atlantic.

Figs 26–30. Porphyra rosengurttii. Reproductive anatomy of fresh blade. Specimen from Cove Island, Stamford Connecticut, USA. Fig. 26. Mixed sorus of mature male and female gametangia, surface view (SV). Fig. 27. Male gametangia, transverse section. Fig. 28. Zygotosporangia in SV. Fig. 29. Young zygotosporangia with trichogynes. Fig. 30. Mature zygotosporangia with residual trichogynes. Scale bars: 10 mm.


J. Brodie et al. The five sequences differed by a single transition at rbcL position 1413, where the specimens from Greece had C and the north-western Atlantic specimens T. Shorter segments (260–1140 bp) from the 30 end of the rbcL gene plus the spacer were sequenced from 21 additional north-western Atlantic specimens. Across the overlapping regions, all were identical to the longer northwestern Atlantic sequences, and all had T at position 1413. Two short rbcL segments (positions 950–1168 and 1360–1467 plus the spacer) were successfully sequenced from a specimen (BM000806061) labelled as ‘401. P. leucosticta’ in Hauck & Richter’s Phykotheka universalis which was collected from Trieste (Adriatic) in 1889. The sequences were identical to the north-western Atlantic P. olivii specimens including T at position 1413. The rbcL–rbcS spacer region was sequenced in two additional specimens from the type location (Gulf of Thessaloniki); these were identical to the other P. olivii specimens. The ITS1 spacer was sequenced from the P. olivii holotype (BM000806050), a second specimen from the type location (BM000806051), plus seven north-western Atlantic specimens. Sequences of

Figs 31–34. Porphyra rosengurttii. Reproductive anatomy of blade and chromosomes. Specimens from Lagos, Spain. Fig. 31. Mature male gametangium in semi-thin transverse section (TS) showing eight tiers four. Fig. 32. Mature zygotosporangium in semi-thin TS showing four tiers two. Fig. 33. Mature zygotosporangium of fresh material showing packages of four, surface view, arrow points to elongated trichogyne. Fig. 34. Chromosomes of male gametes, n ¼ 4. Scale bars: 25 mm (Figs 31–33) and 10 mm (Fig. 34).

20 the two Greek specimens differed from each other by a single bp transition. The ITS1 sequences from the seven north-western Atlantic specimens were identical to each other, but differed from the Thessaloniki specimens by 3–4 bp substitutions. Sequences from a segment near the 30 end of the rDNA SSU gene were obtained for five P. olivii specimens from the Gulf of Thessaloniki, plus one specimen from the north-western Atlantic. All six sequences were identical. Porphyra rosengurttii. A short segment (positions 1156–1467) from the 30 end rbcL gene plus most (72/77 bp) of the rbcL–rbcS spacer was successfully amplified and sequenced from the P. rosengurttii holotype specimen (US55293) collected in 1976 by J. Coll from Pivers Island (Beaufort, North Carolina, USA). Despite triple coverage, the sequence contained one ambiguous base at position 1437, where all three chromatograms had overlapping, equal height C and T peaks. Nearly complete rbcL sequences were obtained from two north-western Atlantic specimens: one (NHA76229, GenBank AY486349, rbcL positions 87–1467 plus partial spacer) from Masonboro Sound (North Carolina, USA) and one (NHA71368, GenBank AF228754, positions 109– 1467 plus partial spacer) from Cove Island Park (Stamford, Connecticut, USA). Most of the rbcL gene was sequenced in two segments (positions 135–1097 and 1181–1467 plus partial spacer) from a specimen collected at Lagos (Malaga, Spain). Partial rbcL (positions 737–1467) plus most of the rbcL–rbcS spacer (72/76 bp) was sequenced from a specimen collected at Cape Brasidas (Gulf of Kavala, Greece). Shorter segments (328–498 bp) from the rbcL 30 end and spacer were sequenced for specimens from Ercolano (Italy), Isla Blanca Park (Port Isabel, Texas, USA) and South Jetty (Port Aransas, Texas, USA). A short rbcL segment (positions 950–1168) was successfully amplified and sequenced from two very old specimens in the Farlow Herbarium (FH). One was collected in 1890 from Porto Mahan (Menorca, Baleares, Spain) and the other in 1906 from Silver Spring (Providence, Rhode Island, USA). Multiple alignment of the rbcL segments from the twelve specimens revealed only a single nucleotide transition located at position 1390. The base at this position was C in the holotype, two specimens from the Gulf of Mexico (Texas, USA), and the specimen from Greece, while it was T in specimens from North Carolina, Connecticut, Italy and Spain. The ambiguous base at position 1437 of the holotype was C in the eight other specimens for which the position was sequenced. While part or all of the rbcL–rbcS spacer was sequenced with the 30 end segment of the rbcL gene

21



Figs 35–38. Historical herbarium specimens from the Mediterranean Sea. Fig. 35. Epiphytic Porphyra atropurpurea material from Venice collected by Olivi, deposited in Venice, Italy. Fig. 36. Isotype material of P. coriacea (two specimens) collected by Zanardini from Venice, Italy and deposited in BM. Fig. 37. Left specimen of Fig. 36, rehydrated and newly spread showing a probably laciniate individual, deposited in BM. Fig. 38. P. rosengurttii material from Venice, Italy, collected under the name of P. coriacea by Titius in the late 1800s; deposited in FH. Scale bars: 2 cm (Figs 35–37) and 5 cm (Fig. 38).


J. Brodie et al. for six specimens, including the holotype, the primer used (rbc–spc) binds to the last 23 positions of the downstream end. Full rbcL–rbcS spacer sequences using primers outside the region were obtained for specimens from Cove Island Park (Stamford, Connecticut, USA), North Carolina (USA), Bracklesham Bay (Sussex, UK), four specimens from Lagos (Malaga, Spain), and a Farlow Herbarium specimen from the Adriatic identified as P. coriacea in the late 19th century by P. Titius. Alignment of full and partial rbcL–rbcS spacer sequences revealed a single nucleotide polymorphism at position 73. Since this was in the primer bind region of the partial sequences, they were eliminated from the alignment. Of the remaining sequences, position 73 was G in the P. coreacea specimen from the Adriatic, the Connecticut and North Carolina specimens, while it was A in specimens from Sussex (UK) and Malaga (Spain). The ITS1 region was sequenced for single specimens of P. rosengurttii from North Carolina (USA), the Gulf of Mexico (Texas, USA), Sussex (UK), Malaga (Spain), and the Gulf of Kavala (Greece), and for two specimens from Connecticut (USA). Sequence similarity over the 306–327 bp region ranged from 92–100%. Only the sequences from the two Connecticut specimens (325 bp) were identical. The North Carolina specimen (327 bp) differed from the Connecticut specimens by a twobp insertion (Fig. 39). The sequences for the specimens from Texas, UK, Spain and Greece were identical in length (306 bp), but differed from each other by 1–4 nucleotide transitions; they differed from the North Carolina and Connecticut specimens by eight short (1–7 bp) insertion/ deletions. Partial rDNA SSU sequences were obtained for five P. rosengurttii specimens from Lagos (Malaga, Spain) plus one from Punta de Galdar (NW Gran Canaria). All six sequences were identical. Molecular comparisons The phylogenetic relationships between P. olivii, P. rosengurttii and other Porphyra species recorded from the North Atlantic were inferred from Bayesian and NJ distance method trees based on trimmed (position 215–1402) and aligned rbcL sequences (Fig. 40). Both Bayesian and NJ

Fig. 39. Condensed alignment of ITS1 between isolates of Porphyra rosengurttii.

22 methods placed P. olivii and P. rosengurttii in a well-supported clade with P. leucosticta and the introduced Asian species P. yezoensis. Pairwise sequence similarity among the twelve Porphyra taxa in the rbcL alignment ranged from 88.4 to 99.1%. Within the clade containing P. olivii and P. rosengurttii, similarity ranged from 91.2 to 97.3%. Pairwise similarity between P. olivii and P. rosengurttii was 97.3% (1298/1333 bp). Similarity between P. olivii and P. leucosticta was 96.6% (1231/1274 bp), and between P. rosengurttii and P. leucosticta it was 96.1% (1224/1274 bp). Discussion The use of molecular, morphological and cytological data has enabled us to resolve the identity of two Porphyra species that were previously confused in the North Atlantic and the Mediterranean. We have described P. olivii as a new species and provided additional morphological and geographical information for P. rosengurttii. It has also been possible to link data between the north-eastern and north-western Atlantic and Mediterranean to show that the distribution of these species is much more widespread than previously thought. We have also been able to confirm that these species are distinct from P. leucosticta, which we know to be widespread in the North Atlantic (Brodie & Irvine, 2003), and that both P. olivii and P. rosengurttii are not recent invaders to the Mediterranean, having been present since at least the late 19th century. Porphyra olivii, P. rosengurttii and P. leucosticta are morphologically very similar but the definition of these species using molecular data has enabled us to determine which morphological characters are useful in their identification (Table 4). The variability in colour of all three species can make identification inconclusive, although the less red or olivaceous tones when living, and the more purplemauve, less bluish tones when dried may help in distinguishing P. leucosticta from the other two species. The blade of all three species can be orbicular in shape, but the tendency of P. olivii to be lanceolate and of P. rosengurttii to be laciniate may also help in their discrimination, as do the more ruffled margins of P. rosengurttii and P. leucosticta compared with P. olivii. However, the development of the male gametangial sori is a character that can be used to distinguish these three species from each other. In P. olivii, the sori form a narrow band that becomes patchy towards the distal end (Fig. 2), whereas in P. rosengurttii they tend to form in a discrete broad zone (Figs 21–24). The scattered sori in P. leucosticta are marginal to submarginal. In P. rosengurttii the tendency of the sori to be long and streaky (Figs 21, 23, 38), and sometimes very pronounced,



23

Fig. 40. Bayesian tree based on trimmed rbcL sequences for Porphyra species from the North Atlantic. Smithora naiadum was used as an outgroup. Node labels are credibility values.

gives the blades a striped appearance, whereas the sori are more diamond to irregular-shaped in the other two species (Figs 2–3, 5; Brodie & Irvine, 2003). The differences in the arrangement of the male and zygotosporangial gametangial packets of the three species can sometimes be useful for identification but interpreting the number of male gametes or zygotospores in a packet can be difficult and may depend on the maturity of the reproductive structures (Brodie & Irvine, 2003). The observation by Coll & Cox (1977) of cells with ‘long projections’ in the type material of P. rosengurttii suggests trichogynes and may indicate material that had not fully developed. This may explain the low number of zygotospores per packet in the original description of the species in comparison with those investigated here. As for many Porphyra species, asexual reproduction seems to be a prominent feature of the life-cycle of P. leucosticta (Holmes & Brodie, 2005), P. rosengurttii (Kapraun & Luster, 1980), and P. olivii (Tsirika et al., 1997; Orfanidis, 2001). In P. leucosticta, archeospores have been observed in different strains of field-collected thalli (Berthold, 1882; Dangeard, 1931; Coll &

de Oliveira Filho, 1976; Gargiulo et al., 1994) and asexual reproduction has also been observed in cultured individuals of this species collected from Germany (Kornmann, 1961) and from southern England (Holmes & Brodie, 2005). Archeospores and neutral spores were observed in P. olivii from Thessaloniki. The induction of these spores and zygotospores appears to be dependent on photoperiod and temperature (Orfanidis, 2001, as P. leucosticta; this study). Orfanidis (2001) reported archeospore formation on blades under 15 C and 20 C SD conditions. In the same culture under LD conditions, spores identified as zygotospores were reported to germinate either uni- or bipolarly. Orfanidis (2001) interpreted this by suggesting that what he had called zygotospores might have been a mixture of zygotospores and asexual spores. Indirect evidence from culture observations of the spores from a single thallus of P. rosengurttii from Lagos (this study) also points to the existence of asexual reproduction in Mediterranean material. The release of two size classes of spores, which germinate either into blades or conchocelis early in the season, followed later by spores of one size, which germinate only

J. Brodie et al.

24

Table 4. Field characters of the blade phase of N. Atlantic to Mediterranean monoecious Porphyra leucosticta, P. rosengurttii and P. olivii. P. leucostictaa

Species Colour

Brown-mauve

Brownish-red, olivaceous to violaceous

Shape

Elliptical, oval, oblong, linear or irregular, occasionally falcate, elongate, umbilicate; not usually laciniate Marginal to submarginal, in distal part of blade

(Sub)orbicular, broad obovate, ovate to reniform; laciniate plants common

Diamond shaped to irregular, scattered Epiphytic

(Broad) irregular to many streaks

Reproductive sori

Form of male gametangia Epiphytic/epizoic/epilithic


P. rosengurttii

Presence on shore Level on shore a

March to Sep., occasionally to Dec. Mid to low intertidal

Discrete broad zone

Mostly epilithic, epizoic, sometimes epiphytic Nov. to May Mid to upper intertidal

P. olivii sp. nov. Brownish red to olivaceous or violaceous (Sub)orbicular to obovate, not laciniate

From base to top, extending sub-marginally towards distal end Diamond shaped to irregular to streaky Epiphytic Dec. to April Shallow subtidal

data from Brodie & Irvine (2003).

into conchocelis, suggests seasonal regulation of spore type formation or germination. In P. rosengurttii from the western Atlantic, the description of monospores with both unipolar and bipolar germination patterns also suggests two types of spores. Thus, it is possible that, under certain conditions of temperature and photoperiod, both spore types develop on the same thallus. The development and function of different spore types in this species complex need further investigation. The karyological differences observed in P. leucosticta may be partly attributable to misidentification of the species, as it is similar to P. rosengurttii and P. olivii, or to the instability of chromosome numbers within individual Porphyra species (see Brodie & Irvine 2003). Geographical strains of P. leucosticta appear to be either sexual, asexual, with n ¼ 3 or n ¼ 4 or n ¼ 3–4 (Coll & de Oliveira Filho, 1977; Yabu, 1978; Kapraun & Freshwater, 1987; Kapraun et al., 1991; Lindstro¨m & Cole, 1992, 1993; Gargiulo et al., 1994; Holmes & Brodie, 2005; Bartsch, pers. obs.). In southern England, one population of P. leucosticta had 3 chromosomes while another had 4 (Holmes & Brodie, 2005). P. olivii is biphasic and has unusually large chromosomes with a haploid chromosome number of n ¼ 2 (Figs 15, 17), while P. rosengurttii from southern Spain has 4 chromosomes in most counts, but occasionally 3 or 2 chromosomes (Fig. 34). The other Mediterranean leucosticta-like species, P. atropurpurea, was reported to have n ¼ 6 (Gargiulo et al., 1999), but no picture was provided and the identity of this material needs checking. Differences in chromosome number within a single species were also observed in P. dioica: individual thalli mostly had

5, but sometimes only 3 chromosomes (Holmes & Brodie, 2004). Kapraun & Freshwater (1987) proposed that the basic haploid chromosome number for North Atlantic Porphyra species was four. They speculated that karyotypes with n ¼ 3 evolved by deletion of the shortest chromosome, number 4. However, Kapraun et al. (1991) also proposed that the karyotype of n ¼ 3 in P. leucosticta and P. spiralis var. amplifolia evolved as a result of a fusion process rather than a deletion event. Aspects of the ecology can also be useful in differentiating these species (Table 4). Porphyra olivii and P. rosengurttii are winter–spring annuals, whereas P. leucosticta is a spring–summer annual. Porphyra olivii and P. leucosticta are epiphytic on other algae whereas P. rosengurttii is typically epilithic. All three occur at different shore levels: P. olivii is found in the shallow subtidal, P. rosengurttii in the mid to upper shore and P. leucosticta mid to lower shore. Specimens from different localities show slight variation but more data are required to determine whether these differences are significant. Porphyra olivii tends to be slightly longer and narrower in the USA compared with the Mediterranean. There are morphological and ecological differences between different locations for P. rosengurttii but the main difference appears to be in the arrangement of the reproductive packets. The known distributions of P. olivii and P. rosengurttii also differ. Both species occur along the north-western Atlantic coast and the Mediterranean, whereas the latter is also reported from the south coast of England and the Canaries. P. olivii has been reported from further north than P. rosengurttii along the north-western Atlantic coast and there are fewer records from the


Cryptic diversity in the Porphyra leucosticta complex Mediterranean for the former species. As these might have been confused with each other, or with P. leucosticta and other species of the ‘leucosticta complex’, biogeographical data are still fragmentary. Ecophysiological data for P. olivii (Orfanidis & Haritonidis, 1996; Orfanidis, 2001) indicate a physiological tolerance for a wide distribution on temperate coasts of the world’s oceans. The distribution for P. leucosticta is reported to be throughout much of the North Atlantic and Mediterranean (see Brodie & Irvine, 2003) but this requires confirmation because of the confusion with related species. It is possible that P. leucosticta is not present in the Mediterranean, but that older reports refer to P. rosengurttii. It is probable that Hamel’s (1924) two forms of P. leucosticta, f. atlantica and f. mediterranea, represent P. leucosticta and P. rosengurttii, respectively. The Atlantic material from Cherbourg grew predominantly epiphytically in the spring and summer and matches P. leucosticta, while the striped male gametangial sori of the Mediterranean material and its occurrence in winter to spring concur with our observations of P. rosengurttii. Feldmann (1939) also described a P. leucosticta population at Banyuls (France) in the Mediterranean as epilithic, which is typical for P. rosengurttii. He also denoted the exclusively winter to spring appearance of Mediterranean populations as ‘P. leucosticta’. It is also possible that Thuret’s original description of P. leucosticta (Le Jolis, 1863) comprised two species, namely P. leucosticta and P. rosengurttii. Thuret pointed out that the species grew on stones and on other algae in the lowest eulittoral and that it disappeared before the end of spring, characters that tend to fit P. rosengurttii more than P. leucosticta. Even Thuret’s description of male gametangia as dispersed around the blade in distinct elongated but irregular specks (Le Jolis (1863) p. 101 ‘. . . les anthe´ridies sont disperse´es autour de la fronde en petites taches distinctes, de forme allonge´e,. . .’) may be attributed to both species; the description fits the form of male gametangia of P. leucosticta, but Thuret also reported that sometimes they are numerous, forming parallel whitish streaks (Le Jolis (1863) p. 101 ‘. . . mais tre`s irre´gulie`res, quelquefois tre`s nombreuses et formant comme des stries paralle`les,. . .’), which may describe the prominent male gametangial arrangement that was observed in Mediterranean P. rosengurttii thalli during this study. Specimens of P. rosengurttii from England, the Canaries and the Mediterranean represent the first records of this species for the eastern Atlantic/ Mediterranean region. While Canaries and Mediterranean material had been misidentified and confused with P. leucosticta and/or

25 P. umbilicalis (e.g. Lopez-Figueroa & Niell, 1989; Flores-Moya et al., 1997; P. rosengurttii as P. umbilicalis: BCM, Reg. 4520 and 4521, coll. R. Haroun), and a recent checklist of the marine plants of the Canary Islands only lists P. carolinensis, P. leucosticta and P. umbilicalis (Haroun et al., 2002), there are several possible reasons why it was overlooked in the UK. It occurs fairly high up on the shore on rocks, and its small, rosette form may have meant that it was confused with P. umbilicalis. Indeed it was collected as such, and only when it was floated out in water were the ‘leucosticta’ sori visible. However, the southern UK occurrence could also reflect warming conditions along the south coast of England. Porphyra rosengurttii appears to be a warmer water species than P. leucosticta, but comparative experimental evidence is still lacking. Porphyra olivii has not been recorded from the UK but there are other unidentified species in the UK flora that may represent this species (Robba et al., 2006). Evidence from rbcL, ITS and rDNA SSU from freshly collected and historical herbarium material (e.g. material from Trieste 1889) indicates that P. olivii from the north-western Atlantic is the same species as that in Greece, although there was notable variation within the rbcL–rbcS spacer region. The presence of a transition at position 73 in the spacer was observed for the first time in Porphyra (Brodie, pers. obs.). In a previous study (Brodie et al., 1996), the presence of a 1 bp difference between specimens in the spacer region was interpreted as indicative of two separate species (P. purpurea and P. dioica) supported by additional morphological and molecular data (Brodie & Irvine, 1997, Klein et al., 2003). The difference in the rbcL for these species was450 bp (94.9%) (Klein et al., 2003, West et al., 2005) compared with 1 bp in P. rosengurttii in our study. All the partial rDNA SSU sequences for this species in this study were identical, although they were obtained only from Spain and the Canaries. Our results compare favourably with those for P. suborbiculata (Broom et al., 2002), with which P. carolinensis and P. lilliputiana were considered synonymous. For example, ITS1 sequence similarity over 279 bp among samples of P. suborbiculata, P. carolinensis and P. lilliputiana was 94.6–100% and for P. rosengurttii 92–100% over 306–327 bp in this study. The slight molecular variation between specimens of P. rosengurttii suggests variation in different parts of the geographical area and studies are required to investigate this further. We were unable to resolve the identity of type material of P. coriacea (Figs 36, 37) using molecular markers. Even after rehydration (Fig. 32), there was no


J. Brodie et al. clear evidence on form and reproduction type. However, the material identified as P. coriacea by Titius (Fig. 38) proved to be P. rosengurttii. At the outset of this study two additional rbcL sequences from P. rosengurttii were held in GenBank, both from North Carolina. One (U04042) is a partial sequence that matches our North Carolina specimen, while the other (AF452442) is different (16 bp) and probably represents a misidentification. The relationship of P. atropurpurea to the species in this paper remains unresolved and, in the absence of definitive type material, is likely to remain so. We have been unable to examine the material in Olivi’s herbarium (Fig. 35) in person, but observations by C. Jarvis (pers. comm.) indicate that there are no male gametangial patches characteristic of ‘leucosticta’. The original account describes a neat white line along the margin and a dark purple colour. Feldmann (1939) interpreted this as male specimens of P. umbilicalis, but the neat white line might also be a developmental stage of P. olivii, with distinct marginal male gametangia from base to the top. Furthermore, although this material is labelled by Olivi as ‘Ulva atro-purpurea’, this is not designated as the type of the species, nor does it resemble the illustrations of this species in Olivi’s original description (Olivi 1794, p. 153). Although the original description denotes attachment to stones, the material illustrated in Fig. 35 appears to be several epiphytic individuals and bears some resemblance to P. olivii (compare Fig. 5), which it may represent, although we cannot be certain.

Acknowledgments We are indebted to many people for this work. We thank Jim Norris (US), Donald H. Pfister (FH), Enrico Ratti (Museo Storia Naturale, Venice) and Charlie Jarvis (BM) for examining Olivi’s Ulva atro– purpurea, and Jenny Bryant (BM) for access to historical specimens. We thank Wilson Freshwater for specimens of P. rosengurttii from Masonboro Sound, Charles Yarish (UConn) for collections from Long Island Sound and for advice on collecting in New York, Connecticut, Rhode Island and southern Massachusetts, Mary Holmes for collections of P. leucosticta from the UK, Ian Tittley (BM) for P. rosengurttii from Bracklesham Bay, Jose Aguilera and Felix Lopez-Figueroa (University of Malaga) for P. rosengurttii from Lagos, Spain, and Ricardo Haroun for this species from the Canary Islands. Abe Tucker, Kelly Vining and Sheri Whipple (UNH Hubbard Center for Genomic Science) helped with sequencing and Anita Klein (UNH) gave advice on molecular work. We also thank Anneke Breeman (University of Groningen) for hospitality and

26 working facilities during S.O.’s stay in her laboratory (European Commission HCM grant), Dr L. P. Pijnacker and Miss M. A. Ferwerda (University of Groningen) for help with karyological studies of P. olivii, Konstantinia Nakou (NAGREF-FRI) for assistance in herbarium preparation of Greek samples, and Regine Schumacher (AWI) for assistance with culture work, karyology and photography of P. rosengurttii from Spain. AWI supported J.B.’s work in Bremerhaven, and Linda Medlin and Thomas Mock provided lab facilities and assistance. We also thank Norman Robson (BM) for assistance with the Latin diagnosis.

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