Phylomineralogy of the Coralline red algae: Correlation of skeletal mineralogy with molecular phylogeny

Phytochemistry 81 (2012) 97–108

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Phylomineralogy of the Coralline red algae: Correlation of skeletal mineralogy with molecular phylogeny A.M. Smith a,⇑, J.E. Sutherland b,1, L. Kregting a,2, T.J. Farr c,3, D.J. Winter d a

Department of Marine Science, University of Otago, P.O. Box 56, Dunedin 9054, New Zealand Department of Biochemistry, University of Otago, P.O. Box 56, Dunedin 9054, New Zealand c National Institute of Water and Atmospheric Research (NIWA), Private Bag 14901, Kilbirnie, Wellington 6241, New Zealand d Allan Wilson Centre for Molecular Ecology and Evolution, Department of Zoology, University of Otago, P.O. Box 56, Dunedin 9054, New Zealand b

a r t i c l e

i n f o

Article history: Received 25 October 2011 Received in revised form 10 June 2012 Available online 13 July 2012 Keywords: Corallinaceae Hapalidiaceae Sporolithaceae Sporolithales Corallinales Corallinophycidae Phylogeny Carbonate mineralogy Aragonite Calcite Mg calcite

a b s t r a c t The coralline algae in the orders Corallinales and Sporolithales (subclass Corallinophycidae), with their high degree of mineralogical variability, pose a challenge to projections regarding mineralogy and response to ocean acidification. Here we relate skeletal carbonate mineralogy to a well-established phylogenetic framework and draw inferences about the effects of future changes in sea-water chemistry on these calcified red algae. A collection of 191 coralline algal specimens from New Zealand, representing 13 genera and 28 species, included members of three families: Corallinaceae, Hapalidiaceae, and Sporolithaceae. While most skeletal specimens were entirely calcitic (range: 73–100 wt.% calcite, mean 97 wt.% calcite, std dev = 5, n = 172), a considerable number contained at least some aragonite. Mg in calcite ranged from 10.5 to 16.4 wt.% MgCO3, with a mean of 13.1 wt.% MgCO3 (std dev = 1.1, n = 172). The genera Mesophyllum and Lithophyllum were especially variable. Growth habit, too, was related to mineralogy: geniculate coralline algae do not generally contain any aragonite. Mg content varied among coralline families: the Corallinaceae had the highest Mg content, followed by the Sporolithaceae and the Hapalidiaceae. Despite the significant differences among families, variation and overlap prevent the use of carbonate mineralogy as a taxonomic character in the coralline algae. Latitude (as a proxy for water temperature) had only a slight relationship to Mg content in coralline algae, contrary to trends observed in other biomineralising taxa. Temperate magnesium calcites, like those produced by coralline algae, are particularly vulnerable to ocean acidification. Changes in biomineralisation or species distribution may occur over the next few decades, particularly to species producing high-Mg calcite, as pH and CO2 dynamics change in coastal temperate oceans. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction At first glance it seems that a photosynthetic marine organism would neither need nor want a skeleton. The energy needed to produce biominerals could instead contribute to growth and reproduction, and indeed the presence of skeletal carbonate may interfere with light-gathering and flexibility. But apparently the benefits of strength, protection, and/or calcium storage outweigh the costs: ⇑ Corresponding author. Tel.: +64 3 479 7470; fax: +64 3 479 8336. E-mail addresses: [email protected] (A.M. Smith), j.sutherland@auckland. ac.nz (J.E. Sutherland), [email protected] (L. Kregting), tracy.farr@royalsociety. org.nz (T.J. Farr). 1 Present address: School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand. 2 Present address: School of Planning, Architecture and Civil Engineering, Queens University of Belfast, Northern Ireland BT7 1NN, United Kingdom. 3 Present address: The Royal Society of New Zealand, P.O. Box 598, Wellington 6140, New Zealand. 0031-9422/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.phytochem.2012.06.003

perhaps 5–6% of marine macroalgae calcify (e.g., Ginsburg et al., 1971) and about 1000 species of calcified marine algae occur from the tropics to the poles throughout the euphotic zone, in a variety of different habitats (Nelson, 2009). While calcified brown (class Phaeophyceae) and green (phylum Chlorophyta) algal species occur, it is in the class Florideophyceae (phylum Rhodophyta) that calcifying macroalgae reach their greatest diversity and latitudinal range (Bosence, 1991). Both crustose and erect calcified red algae are near-ubiquitous in temperate intertidal and subtidal environments, where they provide settlement substrates, structure and shelter on rocky substrates. Rolling pavements of free-living coralline algae (rhodoliths or maerl) form widespread carbonate sedimentary environments known for their high biodiversity (Nelson, 2009). Crustose coralline algae play important roles in tropical reefs, both cementing corals together and producing substantial amounts of carbonate themselves: up to 10 kg/m2/y on the Great Barrier Reef (Chisholm, 2000; Vroom, 2011). In the Antarctic Ross Sea, crustose corallines

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A.M. Smith et al. / Phytochemistry 81 (2012) 97–108

may cover up to 60% of the seafloor, down to a depth of 26 m (Zaneveld and Sanford, 1980). Individual crustose temperate coralline algae may live several centuries; they thus have considerable utility as paleoclimatic records (e.g., Halfar et al., 2008; Gamboa et al., 2010). Accelerating changes in sea-water chemistry, particularly decreasing pH and carbonate concentrations in surface waters, have been shown to affect marine calcifying organisms in a variety of ways, mostly deleterious. As saturation levels fall, the calcium carbonate polymorphs will be affected differently: aragonite is likely to dissolve before calcite, depending on the degree of Mg substitution in calcite (calcite with Mg substitution greater than about 12 wt.% MgCO3 is more soluble than aragonite; Andersson et al., 2008). Initial studies suggest that elevated pCO2 and consequent lowered pH adversely affects recruitment, growth and calcification in some species among the Rhodophyta (e.g., Gao et al., 1993; Kuffner et al., 2008). Projections of long-term effects of changes in sea-water carbonate chemistry on algal communities depend on knowing the carbonate mineralogy of each species – an impossible task. The ability to generalise, and thus to reasonably predict the mineralogy of whole groups, is essential. The simplest generalisation, commonly made, is that among red algae, members of the orders Nemaliales and Peyssonneliales precipitate an extracellular skeleton made of either aragonite needles or needle aggregates (Lowenstam, 1955; James et al., 1988), whereas calcareous species in the Corallinales and Sporolithales produce high-Mg calcite prisms by calcification of the cell wall (see, e.g., Borowitzka et al., 1974; Flajs, 1977; Lowenstam and Wiener, 1989), and that no calcareous alga precipitates a mixed or bimineral skeleton (Ginsburg et al., 1971). In fact the situation is more complex: Mg content in at least some coralline algae may vary as a function of sea-water temperature and perhaps other environmental parameters (Ries, 2006; Kamenos et al., 2008), or due to seasonal changes in growth rate (Kolesar, 1978). At least some species of coralline algae produce calcitic skeletons, but repair damage to their substrate attachment with granules of aragonite (Walker and Moss, 1984). Milliman et al. (1971) identified a shortfall of Mg in calcite – some algal skeletons appear to contain more Mg than can be accounted for by magnesian calcite alone; Weber and Kaufman (1965) found the mineral brucite (Mg(OH)2) in Goniolithon. About a dozen studies of carbonate mineralogy in red algae have been published, offering data on 219 specimens in 135 species in 29 genera (Table 1). The Peyssonneliales are generally

entirely aragonitic (17 specimens, 9 species, 3 genera have been studied), though Flajs (1977) stated that at least some members of this family may form secondary layers of aragonite atop a primary layer of more or less pure calcite. The Nemaliales (32 specimens, 24 species, 3 genera) have so far been found to be entirely aragonitic, though only about 10% of known species have been studied. We know of no published data on skeletal mineralogy of the Gracilariales. The orders Sporolithales and Corallinales in the Corallinophycideae, in contrast, produce mainly calcite, with a wider degree of mineralogical variability, though the wider observed variation may be due to greater availability of data (169 specimens, 101 species, 22 genera). Members of the family Hapalidiaceae (48 specimens, 27 species, 4 genera) produce calcite with Mg content ranging from 4 to 17 wt.% MgCO3 (mean = 10.2, n = 31). Species in the family Corallinaceae (121 specimens, 74 species, 18 genera) appear to construct most skeletons from high-Mg calcite, but some incorporate a small amount of aragonite. Almost all specimens range from 95 to 100 wt.% calcite (mean = 96, n = 55), though Medakovic´ et al. (1995) reported on two specimens of Pseudolithophyllum expansum with 85% aragonite. Coralline algal calcite ranges from 9 to 25 wt.% MgCO3 (mean = 17, n = 35) with the majority of samples containing Mg greater than 12 wt.% MgCO3. These data are not entirely satisfactory. It is quite clear that species identifications can be problematic; Adey and MacIntyre (1973) documented many examples of misidentification, unjustified simplification, and inaccuracy in studies of the crustose coralline algae, and such errors clearly did not end there (see, e.g., Woelkerling and Nelson, 2004; and our corrections to nomenclature made in Tables 1 and 2). A further difficulty is that a variety of methods have been used for mineralogical analysis (usually X-ray diffractometry but some staining and titration), and a range of units reported. Often mineralogy is reported only in a descriptive way without quantitative data; only 90 specimens (45%) have quantitative mineralogical data reported. In all taxa, coverage of the data is skimpy: ca. 10– 15% of species in the Corallinales and Nemaliales have some published mineralogical data available, and only ca. 5% of the Sporolithales. No published mineralogical data are available for any species in the Rhodogorgonales. The coralline algae (orders Corallinales and Sporolithales), with their high degree of mineralogical variability, pose the greatest challenge to projections regarding mineralogy and response to ocean acidification. Fragoso et al. (2010) noted the possibility of phylogenetic control on small biomineralogical variations within the

Table 1 Skeletal carbonate mineralogy of calcifying red algae (Rhodophyta, Florideophyceae), based on 213 reported specimens (Clarke and Wheeler, 1922; Bass-Becking and Galliher, 1931; Lowenstam, 1955; Borowitzka et al., 1974; Flajs,1977; Kolesar, 1978; James et al., 1988; Kerkar, 1994; Medakovic´ et al., 1995; Mannino, 2003; Kamenos et al., 2008; Fragoso et al., 2010). Taxonomic names have been updated according to AlgaeBase. Supplementary data available online. Orders

Families

Genera

Species

Specimens

79 65

163 124

Hapalidiaceae

20 16: Amphiroa, Arthrocardia, Bossiella, Calliarthron, Corallina, Clathromorphum, Hydrolithon, Jania, Lithophyllum, Lithothrix, Metagoniolithon, Metamastophora, Neogoniolithon, Pneophyllum, Spongites, Titanoderma 4: Lithothamnion, Melobesia, Mesophyllum, Phymatolithon

14

Sporolithaceae

1 1: Sporolithon

Galaxauraceae Liagoraceae

8 4: Actinotrichia, Dichotomaria, Galaxaura, Tricleocarpa 4: Liagora, Ganonema, Titanophycus, Yamadaella

Peyssonneliaceae

3 3: Peyssonnelia, Polystrata, Sonderopelta

Corallinales Corallinaceae

Sporolithales Nemaliales

Peyssonneliales

wt.% Calcite

wt.% MgCO3

15–100 mean = 96.2, n = 55

9.0–25.2 mean = 16.8, n = 30

39

100

3.8–17.0 mean = 10.2, n = 31

1 1

1 1

100

13.1 (n = 1)

15 8 7

32 18 14

0 0

n.a. n.a.

9 9

17 17

0

n.a.

99

A.M. Smith et al. / Phytochemistry 81 (2012) 97–108

Table 2 Skeletal carbonate mineralogy of 93 species of calcifying coralline algae (Rhodophyta: Florideophyceae: Corallinophycidae), from a total of 355 specimens (see references for Table 1, and Supplementary data available online). (T) marks type species of genus. Currently accepted species (AlgaeBase)

Published Genus

Published Species

Corallinales: Corallinaceae Amphiroa anceps Amphiroa anceps Amphiroa anceps Amphiroa beauvoisii Amphiroa beauvoisii Amphiroa capensis Amphiroa cryptarthrodia Amphiroa ephedraea Amphiroa fragilissima

Amphiroa Amphiroa Cheilosporum Amphiroa Amphiroa Amphiroa Amphiroa Amphiroa Amphiroa

anceps dilatata anceps beauvoisii mexicana capensis cryptarthrodia ephedraea fragilissima

Mineralogy

wt.% Calcite

wt.% MgCO3 in calcite

Sources

6 1 1 1 1 1 3 1 3

MgC C C C MgC C MgC C MgC

100

13–16

100

16–20

100

18–20

nodulosa rigida

1 3

C MgC

16–20

Amphiroa Cheilosporum Arthrocardia Arthrocardia Arthrocardia Amphiroa Bossiella Calliarthron

tribulus palmatum sp. 1 sp. 2 sp. dorbigniana orbigniana cheilosporoides

1 1 2 5 1 1 1 1

MgC C MgC MgC C MgC MgC MgC

99– 100 100

19.3

100 100

13–15 12–14

This study Flajs (1977) Flajs (1977) Flajs (1977) Fragoso et al. (2010) Flajs (1977) Medakovic´ et al. (1995) Flajs (1977) Clarke and Wheeler (1922), Kerkar (1994) and Flajs (1977) Flajs (1977) Flajs (1977) and Medakovic´ et al. (1995) Clarke and Wheeler (1922) Flajs (1977) This study This study Flajs (1977) Bass-Becking and Galliher (1931) Fragoso et al. (2010) Fragoso et al. (2010)

Amphiroa Amphiroa

Amphiroa

tuberculosa

1

C

Calliarthron

tuberculosum

5

MgC

Cheilosporum

vertebrale

1

C

Phymatolithon

compactum

1

MgC

Corallina

cuvieri

2

MgC

Corallina elongata Corallina officinalis (T)

Corallina Corallina

mediterranea officinalis

1 4

C MgC

Corallina officinalis (T) Corallina officinalis var. chilensis Corallina pilulifera Corallina vancouveriensis Corallina sp.

Corallina Corallina

officinalis chilensis

13 1

MgC C

Corallina Corallina

pilulifera vancouveriensis

1 1

C MgC

Corallina

sp.

Lithophyllum Lithothamnion Melobesia Lithophyllum Lithophyllum

craspedium craspedium farinosa onkodes pachydermum

1 1 1 1 3

MgC MgC C MgC MgC

Goniolithon Jania

reinboldii longifurca

1 4

C MgC

Corallina Haliptilon Haliptilon

1 1 3

C MgC MgC

Haliptilon Jania Jania

lenormandiana gracile rosea ‘bottlebrush’ rosea ‘feather’ rubens corniculata

3 4 2

Jania Cheilosporum Corallina Jania Jania Corallina Lithophyllum Lithophyllum Lithophyllum

sagittata spectabilis squamata tenella verrucosa granifera antillarum byssoides lichenoides

7 3 1 1 8 1 1 2 2

Amphiroa nodulosa Amphiroa rigida Amphiroa tribulus(T) Arthrocardia palmata Arthrocardia sp. Arthrocardia sp. Arthrocardia sp. Bossiella orbigniana Bossiella orbigniana Calliarthron cheilosporoides (T) Calliarthron tuberculosum Calliarthron tuberculosum Calliarthron tuberculosum Clathromorphum compactum Corallina cuvieri

Hydrolithon craspedium Hydrolithon craspedium Hydrolithon farinosum Hydrolithon onkodes Hydrolithon pachydermum Hydrolithon reinboldii Jania longifurca Jania pusilla Jania rosea Jania rosea Jania rosea Jania rubens (T) Jania rubens var. corniculata Jania sagittata Jania spectabile Jania squamata Jania tenella Jania verrucosa Jania virgata Lithophyllum antillarum Lithophyllum byssoides Lithophyllum byssoides

Specimens

13

MgC/A

Flajs (1977) 100

11–12

Kolesar (1978) Flajs (1977)

100

10.9

99– 100

16–20

100

12–15

Clarke and Wheeler (1922) Borowitzka et al. (1974) and Flajs (1977) Flajs (1977) Bass-Becking and Galliher (1931), Flajs (1977) and Medakovic´ et al. (1995) This study Flajs (1977) Flajs (1977) Fragoso et al. (2010)

89– 100 100 100

11–14

This study

19.6 15.9

100 100

18.2 15–25

Clarke and Wheeler Clarke and Wheeler Flajs (1977) Clarke and Wheeler Clarke and Wheeler

98– 100

16–20

Flajs (1977) Medakovic´ et al. (1995)

100

13–15

Flajs (1977) Fragoso et al. (2010) This study

MgC MgC C

100 100

13–15 9.0

This study Kerkar (1994) and Flajs (1977) Flajs (1977)

MgC MgC C MgC MgC C MgC MgC MgC

100 100

12–15 15.0

100

12–16

100 98–99 98

16.4 13–15 16–20

This study Flajs (1977) and Kerkar (1994) Flajs (1977) Fragoso et al. (2010) This study Flajs (1977) Clarke and Wheeler (1922) Mannino (2003) Medakovic´ et al. (1995)

(1922) (1922) (1922) (1922)

(continued on next page)

100

A.M. Smith et al. / Phytochemistry 81 (2012) 97–108

Table 2 (continued) Currently accepted species (AlgaeBase)

Published Genus

Published Species

Specimens

Mineralogy

wt.% Calcite

wt.% MgCO3 in calcite

Sources

Lithophyllum carpophylli

Lithophyllum

carpophylli

15

MgC/A

12–16

This study

Lithophyllum congestum Lithophyllum corallinae

Lithophyllum Lithophyllum

daedaleum corallinae

1 6

MgC MgC/A

88– 100 100 80– 100

19.0 12–15

Clarke and Wheeler (1922) This study

cystoseirae aff.

Melobesia Lithophyllum

cystoseirae aff. frondosum

1 1

C MgC

incrustans

Lithothamnion

incrustans

3

MgC

incrustans

Lithophyllum

incrustans

1

C

Lithophyllum

intermedium

1

MgC

100

16.6

Clarke and Wheeler (1922)

Lithothamnion

kaiserai

1

MgC

100

15.3

Clarke and Wheeler (1922)

Pseudolithophyllum

orbiculatum

1

C

Lithophyllum Dermatolithon

pallescens pustulatum

1 1

MgC C

100

15.5

Clarke and Wheeler (1922) Flajs (1977)

Lithophyllum

pustulatum

7

MgC/A

95– 100

12–15

This study

Lithophyllum Lithophyllum Lithophyllum

molluccense tamiense racemus

1 1 5

MC MgC MgC/A/D

20.0 16–20

Lithophyllum racemus Lithophyllum riosmenae Lithophyllum stictaeforme Lithophyllum tortuosum Lithophyllumtortuosum Lithophyllum tortuosum Lithophyllum sp.

Lithothamnion Lithophyllum Lithophyllum

racemus riosmenae stictaeforme

2 1 10

MgC MgC/A MgC/A

100 90– 100 100 98 90– 100

Lithophyllum Lithothamnion Tenarea Lithophyllum

tortuosum tortuosum undulosa sp. 1

1 2 2 4

C MgC MgC MgC/A

Lithophyllum sp. Lithothrix aspergillum (T) Mastophora pacifica Metagoniolithon radiatum Metagoniolithon stelliferum Metamastophora flabellata Metamastophora flabellata Metamastophora flabellata Neogoniolithon acropetum Neogoniolithon brassicaflorida Neogoniolithon brassicaflorida Neogoniolithon brassicaflorida Neogoniolithon mamillosum Neogoniolithon orthoblastum Neogoniolithon strictum

Lithophyllum Lithothrix

sp. aspergillum

1 1

MgC MgC

Mastophora Amphiroa

pacifica charoides

1 1

MgC C

Amphiroa

stelligera

1

C

Flajs (1977)

Mastophora

hypoleuca

1

C

Flajs (1977)

Mastophora

lamourouxi

1

C

Flajs (1977)

Mastophora

plana

1

C

Flajs (1977)

Goniolithon

acropetum

1

MgC

100

19.2

Clarke and Wheeler (1922)

Goniolithon

frutescens

2

MgC

100

13.8

Neogoniolithon

brassica-florida

2

MgC/A

97–99

13.1

Clarke and Wheeler (1922) and Flajs (1977) This study

Neogoniolithon

notarisii

1

C

Flajs (1977)

Neogoniolithon

mamillosum

1

C

Flajs (1977)

Goniolithon

orthoblastum

1

MgC

100

13.7

Clarke and Wheeler (1922)

Goniolithon

strictum

5

MgC

100

23–25

Neogoniolithon

trichotomum

1

MgC

Clarke and Wheeler (1922) and Flajs (1977) Fragoso et al. (2010)

Lithophyllum

amplexifrons

1

C

Flajs (1977)

Melobesia Pneophyllum Melobesia Lithothamnion Dermatolithon Litholepis

lejolissi aff. fragile zonalis ramulosum laminariae mediterranea

1 1 1 2 1 1

C MgC C C C C

Flajs (1977) Fragoso et al. (2010) Flajs (1977) Clarke and Wheeler (1922) Flajs (1977) Flajs (1977)

Lithophyllum Lithophyllum frondosum Lithophyllum (T) Lithophyllum (T) Lithophyllum intermedium Lithophyllum kotschyanum Lithophyllum orbiculatum Lithophyllum Lithophyllum pustulatum Lithophyllum pustulatum Lithophyllum Lithophyllum Lithophyllum

pallescens

pygmaeum pygmaeum racemus

Neogoniolithon trichotomum Pneophyllum amplexifrons Pneophyllum fragile Pneophyllum aff. fragile Pneophyllum zonale Spongites fruticulosa Titanoderma laminariae Titanoderma

Flajs (1977) Fragoso et al. (2010) 100

10–14

Clarke and Wheeler (1922) Flajs (1977)

Flajs (1977)

5–11 12.7 13–15

Borowitzka et al. (1974) Clarke and Wheeler (1922) Flajs (1977) and Medakovic´ et al. (1995) Clarke and Wheeler (1922) This study This study

100 100 90– 100 100

9–12 16–20 13–16

Flajs (1977) Clarke and Wheeler (1922) Medakovic´ et al. (1995) This study

12.8

Clarke and Wheeler (1922) Fragoso et al. (2010)

100

13.5

This study Flajs (1977)

100

6.8

101

A.M. Smith et al. / Phytochemistry 81 (2012) 97–108 Table 2 (continued) Currently accepted species (AlgaeBase)

Published Genus

Published Species

Lithothamnion

crispatum

Lithothamnion

Specimens

Mineralogy

wt.% Calcite

wt.% MgCO3 in calcite

Sources

1

MgC/A

86

10.8

This study

fornicatum

1

MgC

100

9.3

Clarke and Wheeler (1922)

Lithothamnion

fruticulosum

1

C

Lithothamnion

glaciale

4

MgC

100

11–25

Lithothamnion

nodosum

1

C

100

6.1

Lithothamnion

propontidis

1

C

Lithothamnion

soriferum

1

MgC

100

9.6

Clarke and Wheeler (1922)

Lithothamnion

sp.

C

100

4–12

Melobesia sp. Mesophyllum engelhartii

Melobesia Mesophyllum

sp. engelhartii

1 6

MgC MgC/A

14.4 12–15

Mesophyllum erubescens Mesophyllum erubescens

Lithothamnion Mesophyllum

erubescens erubescens

1 7

MgC MgC/A

17.0 11–13

Clarke and Wheeler (1922) This study

Mesophyllum expansum

Pseudolithophyllum

expansum

7

C

16–20

Lithothamnion

1

C

Flajs (1977) and Medakovic´ et al. (1995) Clarke and Wheeler (1922)

Mesophyllum

phillipi var. funafutiense lichenoides

100 96– 100 100 92– 100 15– 100 100

Clarke and Wheeler (1922) and Flajs (1977) Clarke and Wheeler (1922) This study

1

C

Mesophyllum

macroblastum

12

MgC/A

Mesophyllum

printzianum

31

MgC/A

Mesophyllum

1

MgC/A

Mesophyllum

sp. cf. erubescens sp.

3

MgC/A

Lithothamnion

calcareum

4

Lithothamnion

polymorphum

Phymatolithon

mediterraneum Corallinales: Hapalidiaceae Lithothamnion crispatum Lithothamnion fornicatum Lithothamnion fruticulosum Lithothamnion glaciale Lithothamnion nodulosum Lithothamnion propontidis Lithothamnio ntophiformes Lithothamnion sp.

Mesophyllum funafutiense Mesophyllum lichenoides (T) Mesophyllum macroblastum Mesophyllum printzianum Mesophyllum sp.

11

Flajs (1977) Clarke and Wheeler (1922), Flajs (1977) and Kamenos et al. (2008) Clarke and Wheeler (1922) Flajs (1977)

5.9

Flajs (1977) 73– 100 73– 100 91.8

11–15

This study

11–16

This study

11.9

This study

11–13

This study

MgC

91– 100 100

10–12

1

MgC

100

9.1

Clarke and Wheeler (1922) and Flajs (1977) Clarke and Wheeler (1922)

calcareum

2

MgC

14–24

Phymatolithon

repandum

1

MgC

97– 100 100

Lithothamnion

tenuissimum

1

C

Heydrichia Heydrichia Sporolithon

homalopasta woelkerlingii durum

1 1 11

MgC MgC MgC/A

Sporolithon sp.

Sporolithon

sp.

10

MgC/A

Sporolithon episporum

Sporolithon

episporum

Mesophyllum sp. Phymatolithon calcareum Phymatolithon calcareum Phymatolithon calcareum Phymatolithon repandum Phymatolithon tenuissimum Sporolithales: Sporolithaceae Heydrichia homalopasta Heydrichia woelkerlingii Sporolithon durum

1

Corallinaceae. Recent phylogenetic analyses of the New Zealand Corallinales and Sporolithales (Broom et al., 2008; Farr et al., 2009) allow us to test this idea in detail. Here we take the unusual opportunity to relate skeletal carbonate mineralogy to phylogeny at a very detailed level. To what extent does skeletal composition in the Corallinophycidae vary in relation to phylogenetic position? This is the first study in any taxon in which both skeletal and molecular sequence data have been obtained and analysed from the same specimens, allowing mineralogical data to be assessed in the context of a well-established

11.2

Medakovic´ et al. (1995) and Kamenos et al. (2008) This study Flajs (1977)

MgC

100 100 98– 100 90– 100 100

12.8 14.5 12–15

This study This study This study

11–14

This study

13.1

Clarke and Wheeler (1922)

phylogenetic framework. We use this framework to ask to what extent skeletal composition in the Corallinales and Sporolithales varies with phylogenetic position, and to draw inferences about the effects of future sea-water chemistry on these calcified red algae. These phylogenetic data are also crucial for determining how environmental and life-history traits contribute to the mineralogy of coralline algae. Our data are drawn from multiple algal lineages, and thus do not represent statistically independent samples (Felsenstein, 1985; Garland et al., 2005). Since some of the between-sample differences we report may be the result of

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A.M. Smith et al. / Phytochemistry 81 (2012) 97–108 Chondrus crispus AY119746 76 0.99

Griffithsia antarctica AY295145 94 1

Aglaothamnion callophyllidicola DQ787641 54 1

Antithamnionella sp. A31 DQ787640 83 1

Herpochondria corallinae DQ787646 67

Ceramium japonicum AY178485

1

50 0.91

Ceramium boydenii AY178484

100 1

Campylaephora borealis AY178480 Heydrichia homalopasta

56 0.79

Heydrichia woelkerlingii 65 0.99

100 1

85 1

Sporolithon sp.

100

Sporolithon durum

1

Mesophyllum sp. cf. erubescens

83 0.96

94

100 1

1

76 0.99

Mesophyllum engelhartii

100

Mesophyllum macroblastum

1

68 1

Lithothamnion crispatum Phymatolithon repandum 51 0.99

100

1

95 1

Mesophyllum erubescens

1

79

100 Mesophyllum sp. 1

91 1

71 0.98

100 1

96 1

100 1 93 1

62 1

Arthrocardia sp. 1

Arthrocardia sp. 2 Corallina officinalis 90 0.99

Corallina sp.

99 1 54 0.92

Mesophyllum printzianum

Jania rosea 99 1

Jania sagittata 100 1

Jania verrucosa Mastophora pacifica 100 1

-

Lithophyllum riosmenae

56 0.91

0.93

Neogoniolithon brassica-florida

100 1

83 0.99

Amphiroa anceps

100 1 61

61 0.99

0.97 51 0.92

87 0.95

Lithophyllum sp. 1 100 1 99 1 100 1 100 1

Lithophyllum corallinae Lithophyllum stictaeforme Lithophyllum pustulatum Lithophyllum carpophylli

0.05

Fig. 1. Maximum likelihood tree derived from psbA sequence data. ML bootstrap support (500 replicates) and Bayesian posterior probability values are shown to the left of each clade. Clades containing more than two sequences are collapsed to a triangle: the length of each triangle is proportional to the amount of sequence variation within the clade. Numbers of sequences in each clade are indicated to the right of the clade name. Family relationships can be found in Fig. 2.

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Fig. 2. Skeletal carbonate mineralogy of 191 algal specimens from 28 species in the Corallinophycidae from around northern New Zealand. All specimens are dominated by high-Mg calcite, but a few specimens, particularly in the genera Lithophyllum and Mesophyllum show aragonite content up to 27%. For individual specimens, see Supplementary data online.

divergent evolutionary history of those samples, we have used Phylogenetic Comparative Methods to detect the presence of such ‘‘phylogenetic signal’’ and to control for this effect in other analyses. 2. Results Of the 487 samples analysed by Farr et al. (2009), 191 could be identified to genus or species level, yielded psbA sequence data,

and were large enough for XRD analysis (Table 2). The 191 specimens represented 28 species in 13 genera. The collections included members of three families in two orders: family Corallinaceae (order Corallinales) (n = 106, 7 genera, 16 species), family Hapalidiaceae (order Corallinales) (n = 62, 3 genera, 8 species), and family Sporolithaceae (order Sporolithales) (n = 23, 2 genera, 4 species). The maximum likelihood tree constructed from the New Zealand specimens used for the mineralogy analysis is shown in

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Fig. 1. The three families are resolved as mutually monophyletic, as has been shown in a number of other analyses (Broom et al., 2008; Farr et al., 2009). Most skeletal specimens were entirely calcitic, but a considerable number (76 specimens) contained a small amount of aragonite. Calcite content ranged from 73 to 100 wt.% calcite, with a mean of 97 wt.% calcite (std dev = 5, n = 172). Mg in calcite ranged from 10.5 to 16.4 wt.% MgCO3, with a mean of 13.1 wt.% MgCO3 (std dev = 1.1, n = 172). While the mineralogy of specimens is broadly as we would expect (mainly high-Mg calcite), there is a surprising degree of variability (Fig 2; see also Supplementary data). This is the first study to find appreciable amounts of aragonite produced by marine coralline algae. Mean calcite content in the family Sporolithaceae was 98.7 wt.% calcite (std dev = 2.86, n = 23), not significantly different from that of the Corallinaceae (mean = 98.3 wt.% calcite, std dev = 3.19, n = 106; t-statistic = 0.119, df = 38, p = 0.9053). Both groups were significantly greater in calcite than the family Hapalidiaceae (mean = 94.6 wt.% calcite, std dev = 6.86, n = 62; t-statistics = 4.073 and 4.594 respectively, df = 92 and 91, p < 0.0001). While there is no clear phylogenetic signal in the data on calcite percentage (Bloomberg’s K = 0.013, p = 0.413), Mg content in calcite was found to have a moderately strong and highly significant phylogenetic signal (Bloomberg’s K = 0.026, p < 0.001). Mg content in calcite was significantly different among all three families. Corallinaceae had the highest mean Mg content at 13.6 wt.% MgCO3 (std dev = 1.02, n = 106). The Corallinaceae were significantly different from the Sporolithaceae (mean = 13.0 wt.% MgCO3, std dev = 0.88, n = 23; t-statistic = 2.589, df = 39, p = 0.0134). The Hapalidiaceae had the lowest Mg content and the greatest mineralogical variability (mean = 12.5 wt.% MgCO3, std dev = 1.13, n = 62) and were significantly different from both the Corallinaceae (t-statistic = 6.330, df = 140, p < 0.0001) and the Sporolithaceae (t-statistic = 2.338, df = 53, p = 0.0232). While carbonate mineralogy among the coralline algae in New Zealand is broadly similar, with almost all species producing 95– 100% calcite with 10–14 wt.% MgCO3 substitution, there are some anomalies that are not randomly distributed with respect to phylogeny (Fig. 1). In particular, most of the variability in the family Corallinaceae occurs in the genus Lithophyllum (n = 57). Without Lithophyllum, the range of calcite in the family is 99–100% calcite, and the range of Mg contents would be similarly small, 11.6– 15.7 wt.% MgCO3 (n = 58). Lithophyllum species increase the range of this family to 80–100 wt.% calcite, and 11.6–16.4 wt.% MgCO3. Calcite content of Lithophyllum species is significantly different from the other species in the Corallinales (t-statistic = 3.969, df = 72, p < 0.0001), as is Mg content (t-statistic = 2.692, df = 110, p = 0.0093). Species in the Sporolithaceae (order Sporolithales) are indistinguishable, mineralogically, from most of the Corallinaceae. It is in the family Hapalidiaceae that the most mineralogical variability occurs. Of the species studied here, Mesophyllum macroblastum and M. printzianum have the widest range of calcite content (each 73–100% calcite); they vary from 11.4 to 14.8, and from 10.5 to 15.8 wt.% MgCO3 in calcite, respectively (n = 12; n = 31). Other Mesophyllum species studied here vary in range from 91 to 100% calcite, and 11 to 15 wt.% MgCO3 in calcite (n = 17). When the phylogenetic signal in the Mg content data is incorporated in a model with latitude and growth-form as predictor variables, only growth-form (p = 0.034) is found to have a significant effect, with crustose forms having significantly less MgCO3. Geniculate coralline algae are erect and branched, with calcified segments linked by uncalcified nodes; the non-geniculate coralline algae which make up the remaining taxa are completely calcified and crustose. Mineralogically, it is the crustose non-geniculate species that are more variable. For all geniculate taxa assessed in the current study (seven species in the subfamily Corallinoideae,

and a single species in the subfamily Lithophylloideae, all within the family Corallinaceae; geniculate taxa are indicated in Fig. 2 with an asterisk), skeletal calcite ranged from 99.5 to 100% calcite (mean = 99.8 wt.% calcite, std dev = 0.83, n = 62). Of 62 specimens examined, only two (one each of Corallina sp. and Arthrocardia sp.) produced less than 100% calcite. In contrast, the non-geniculate crustose algae contained more species with a higher percentage of aragonite, with calcite accounting for 72.8–100% calcite (mean = 96.2 wt.%, std dev = 5.66, n = 150). The difference is statistically significant (t-statistic = 7.683, df = 163, p < 0.0001). The range of MgCO3 in calcite is 11.6–15.7 wt.% for geniculate taxa (mean = 13.4 wt.% MgCO3, std dev = 0.99, n = 61), significantly higher than in the non-geniculate species (mean = 13.0, std dev = 1.19, n = 130; t-statistic = 2.451, df = 136, p = 0.0155). 3. Discussion The data from this study, taken along with published data, increases the number of calcifying red algae for which some mineralogical data is available to a total of 355 specimens in 93 species (Tables 1 and 2). This quantity is a reasonable proportion of the ca. 1000 extant species that we know produce biominerals as part of their thallus. There are thus some generalisations that can be made regarding the skeletal carbonate mineralogy in this taxon. Species in the Nemaliales and Peyssonneliales appear to form their skeletal elements entirely from aragonite, but adopting two different ultrastructural arrangements. Nemaliales, like many calcifying green algae such as Acetabularia, Halimeda and Penicillus (Flajs, 1977) form their skeletons from aragonitic needles (either singly as in Liagora, or in composite aggregates as in Galaxaura; Flajs, 1977). Calcifying genera in the Peyssonneliales, in contrast, form small aragonitic prisms in a mesh of crystals (James et al., 1988) that lie normal to the cell surface, not unlike the calcitic prisms which can be found in some coralline algae (e.g., Mesophyllum and Goniolithon, Flajs, 1977). Other genera in the Corallinales and Sporolithales (e.g., Lithothaminium) produce almost entirely calcite, alternating layers of calcitic needles with layers of calcitic prisms (Flajs, 1977). Almost every species in the Corallinales and Sporolithales can, however, occasionally produce a small amount of aragonite, in some cases up to 20% aragonite. It seems likely that the two specimens of Pseudolithophyllum reported to have only 15% calcite (Flajs, 1977) are misidentified, contaminated, or affected by their environment. In fact, in the absence of detailed sectioning and imaging of specimens which manifest aragonite, it is impossible to separate primary mineralisation from secondary infilling or early diagenesis. Changes in Mg/Ca ratio with local seasonal sea-surface temperature fluctuations have been found in rhodoliths of Lithothamnium glaciale, L. crassiusculum, and Phymatolithon calcareum (Kamenos et al., 2008; Halfar et al., 2000), but on a spatial scale too small to be measured using XRD. Our data (and those culled from the literature) are time-averaged over the life of the alga, and thus can only elucidate broader trends. There was no significant relationship between aragonite-to-calcite ratio or Mg content and latitude (Fig. 3a) found in the coralline algae. Within the New Zealand data, the only subset for which the phylogenetic history of the samples is known, we again found no significant relationship between latitude and Mg content. Interestingly, latitude approaches significance and is in the predicted direction. It is possible that broader geographic sampling of corallines or direct measurement of water temperature would reveal an effect that we have not been able to measure in this study. Water temperature is only loosely represented by latitude, and temperature is, of course, not the only driving force in coralline algal growth, as it operates in concert with degree of light irradiance (e.g., Halfar et al., 2011), but apparently temperature has a greater influence on Mg:Ca ratio (Halfar et al., 2000). If Mg content is re-

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A

B

Fig. 3. Variations in carbonate mineralogy across latitudes A. in rhodophyte calcite (n = 274 specimens) and B. among New Zealand rhodophytes (n = 212 specimens) (negative latitude = south). Data available in Supplementary data online.

lated to growth rate (e.g., Moberly, 1968), however, then perhaps degree of irradiance may have some effect on mineralogy as well. In any case, our data do not demonstrate a broad relationship between latitude and skeletal carbonate mineralogy, in contrast to the strong inverse relationship shown in other species and even among calcareous algae (Chave, 1954). Geniculate coralline algae contain little or no aragonite, compared with those with a non-geniculate life habit. The absence of aragonite in geniculate specimens may be explained by their structure. If aragonite deposition occurs largely during repair of substrate attachment, as suggested by Walker and Moss (1984), it will be present in only small amounts in geniculate specimens, in which the region of substrate attachment is small in relation to overall thallus size. Also, substrate attachment points may have been absent in some of our geniculate specimens. In contrast, non-geniculate algae form spreading crusts that may contain semi-enclosed spaces or incorporate sediment or shell material. It is possible that the aragonite and brucite observed in these specimens is not precipitated by the alga itself, but incorporated into the skeleton during the process of overgrowth. Under present Earth surface conditions, magnesian calcite is metastable, that is, it is thermodynamically unstable but temporarily able to persist due to kinetic factors. The more Mg present in calcite, the more soluble and vulnerable to dissolution or recrystallisation it is. Coralline algal calcite has the highest degree of substitution by Mg in the natural world (Lowenstam and Wiener, 1989), making it consequently among the least stable of carbonate minerals. As anthropogenic CO2 continues to reduce sea-water pH, magnesian calcites will be among the first to dissolve, and the most energetically difficult minerals to produce (Andersson et al., 2008).

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Temperate organisms are particularly vulnerable, as undersaturation of surface sea water is predicted to occur earlier in cool-tocold water (Andersson et al., 2008). Sea water in temperate regions could become undersaturated with respect to calcites with wt.% MgCO3 > 10 by 2050 (Andersson et al., 2008). Biomineralisers that produce high-Mg calcite, in particular, could thus be under considerable chemical pressure within a few decades. It is not unreasonable to expect functional, structural, reproductive or energetic responses from calcifying organisms immersed in water that is undersaturated with respect to their skeletal materials. Evidence has been accruing over the past decade regarding the possible responses of biomineralising organisms to changes in seawater pH and carbonate availability. While some invertebrates (e.g., some decapods, echinoids and gastropods) calcify more rapidly in high-CO2 experimental settings, others (corals, bivalves, pteropods) appear to calcify more slowly or even to cease calcification altogether (e.g., Doney et al., 2009; Ries, 2011). Some bryozoans have been shown to lower the proportion of energy devoted to defensive heterozooids when under chemical stress (Lombardi et al., 2011). Larvae of both echinoids (Clark et al., 2009) and bivalves (Talmage and Gobler, 2009) have been shown to develop more slowly, with lowered survival and higher rates of malformed skeletons in low-pH water. Among algae, a possible response, according to some studies, is the reduction of chemical stress by changing skeletal mineralogy. Calcifying rhodophytes have been shown to be capable of producing calcite with lower Mg content when ambient sea water has a lower Mg content (Stanley et al., 2002; Ries et al., 2006), and it has been claimed that their mineralogy has changed in step with sea-water chemistry throughout the Phanerozoic (Stanley and Hardie, 1998). Ries’ (2010) experiments showed that three Amphiroa species and one Neogonolithon species were able to adjust Mg content of their skeletons depending on the Mg content of ambient sea water. One species of Amphiroa, which produced calcite with 14 to16 wt.% MgCO3 in normal sea water (Mg:Ca ratio 5.2), was able to produce calcite with less than 1 wt.% MgCO3 only four days later in water with Mg:Ca ratio of 1.0 (Ries, 2010). Alongside this mineralogical flexibility, however, is a loss of skeletal organisation in lower Mg:Ca waters. Some organisms immersed in sea-water undersaturated with respect to their preferred skeletal mineral either produce very thin skeletons or none at all (e.g., Fine and Tchernov, 2007). If coralline algae were to respond in this way, presumably the benefits of calcification in providing three-dimensional support and protection from predation would be removed, and these species would become vulnerable to grazers and physical disturbance. Ries (2010), however, found that thinner skeletons were formed in high-Mg sea water, and that as Mg decreased, skeletons became both thicker and more chaotically organised. While the potential response of rhodophytes to increasing sea-water acidity is unclear, and may vary depending on species, it is highly likely that some effect will manifest itself. Within photosynthetic limits, it might be possible for algae under chemical stress to migrate to deeper or warmer waters. Reports from eastern Australia suggesting poleward range shift of habitatforming algae (Eckloniaradiata, Phyllosporacomosa, Durvillaeapotatorum) in response to changing ocean conditions (increasing water temperatures) are the subject of current study (Wernberg et al., 2009). Hall-Spencer et al. (2008), documenting communities along existing gradients of pH at volcanic CO2 vents, report a shift from typical rocky shore profile with abundant calcareous organisms at normal pH, to communities with significant reduction in coralline algal abundance at lowered pH. Temperate intertidal communities would be very different without calcifying rhodophytes. Erect geniculate coralline turfs have been called ‘ecosystem engineers’ because they provide hab-

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itat and food sources for a variety of invertebrates on the shore, generally enhancing biodiversity (Nelson, 2009). Their resistance to wave action and ability to retain moisture during low tide provide crucial resources to small invertebrates (Nelson, 2009). Similarly, crustose coralline algae strengthen the substrate they encrust, incorporating sediment, shells and loose material into it, playing important roles in stabilizing reefs (e.g., Vroom, 2011). They enhance larval settlement and metamorphosis in invertebrates ranging from corals (Morse et al., 1996) to economically relevant molluscs such as abalone (Roberts, 2001). If CO2 emissions continue to increase, and worst-case ocean acidification projections are accurate, it is likely that a succession of changes will occur among marine algae, and indeed other calcifying species. High-Mg calcite skeletons would be among the earliest affected, with aragonitic species to follow. If pH were to become low enough, it might become impossible for any organisms to calcify (Fine and Tchernov, 2007). The final result could well be a temperate rocky shore covered with plants, anemones, jellies and other soft species, with no turfs, reefs, maerls, or phytoliths to provide shelter or substrate variety.

4. Conclusions Previous generalisations regarding the mineralogy of coralline algae have failed to tell the whole story. Skeletal mineralogical data are now available for 93 species, about 10% of those rhodophytes that do calcify. While most species in the Corallinophycidae are formed of Mg calcite, aragonite, whether primary or secondary, occurs in many specimens, forming up to 27% of the biominerals present. A few taxa are particularly variable, especially within the genera Mesophyllum and Lithophyllum. There is a close relationship between mineralogy and growth habit: erect articulated geniculate coralline algae do not generally contain any aragonite; whereas aragonite is sometimes present in crustose non-geniculate species, perhaps because of their greater substrate attachment area. Mg content varies among coralline families. The Corallinaceae had the highest Mg content, followed by the Sporolithaceae. The Hapalidiaceae had the lowest Mg content and the greatest mineralogical variability. Despite the significant differences among families, there is sufficient variation and overlap to prevent the use of carbonate mineralogy as a taxonomic character. In our data we observe no relationship between latitude (a loose proxy for water temperature) and Mg content in coralline algae, contrary to trends observed in other biomineralising taxa (Chave, 1954). Sampling from a wider distribution of specimens, especially in southern latitudes, is necessary to clarify this relationship. Magnesium calcites, such as those produced by the Corallinophycidae, are particularly vulnerable to ocean acidification, especially in temperate waters. Responses that may become manifest in the next few decades could include changes in biominerals that are produced, changes in species distribution (either in terms of latitude or depth), and thinning or loss of skeletal materials. The taxa producing the highest-Mg calcites are among the most vulnerable, as are those which produce aragonite. The variation in skeletal mineralogy observed in this study suggests that the vulnerability of individual coralline species to ocean acidification is likely to vary considerably. Predictions regarding the responses of calcified macroalgae to ocean acidification based on taxonomic position at family or even genus level may be quite misleading. Understanding the interaction of calcified macroalgae with changing ocean pH will require a better knowledge of algal biominerals and the environmental factors that influence them.

5. Experimental Specimens of calcified algae from the subclass Corallinophycidae were collected from around northern New Zealand during a survey of coralline algal biodiversity in the region from 2005 to 2008. Sample collection localities ranged from 29.2°S in the Kermadec Islands to 38.3°S in the central North Island, New Zealand. Water depths ranged from intertidal to 34 m, but were mostly less than 20 m. Vouchers for all specimens were retained, and reference specimens for each species identified have been lodged at WELT (the herbarium at the Museum of New Zealand Te Papa Tongarewa) as part of the National Coralline Algal Reference Collection. Herbarium accession numbers for reference specimens are listed in Appendix 4, Farr et al. (2009). Specimens were identified from anatomical and morphological characters observed in sections prepared using methods detailed by Harvey et al. (2005). Specimens (or subsamples thereof) that had been air-dried, or dried in silica gel, were used for amplification and DNA sequencing of the photosystem II thylakoid membrane protein D1 (psbA) gene as described in Broom et al. (2008). Sequences were aligned in Se-Al v2.0a11 (Rambaut, 1996) and the maximum likelihood tree was estimated under the GTR + I + G model of sequence evolution using PHYML V3.0 (Guindon and Gascuel, 2003). Support under ML was estimated by bootstrapping (500 replicates). MrBayes v3.1.2 (Huelsenbeck and Ronquist, 2001) was used to estimate the Bayesian tree and posterior probability (PP) values for clades. The data were partitioned by codon position, with the GTR + I + G model applied to each partition independently. Two runs of 5 million generations each were started from random trees; trees were sampled every 100 generations. Burnin was assessed as 500,000 generations using Tracer v1.5 (Rambaut and Drummond, 2007). FigTree v1.3.1 (Rambaut, 2006) was used to collapse clades with more than one member. Eight non-coralline taxa were included as outgroups. GenBank accession numbers are listed by Farr et al. (2009) in their Appendix 3. Skeletal carbonate mineralogy was analysed for specimens for which we had determined psbA sequences, and which therefore could be mapped onto the phylogenetic tree. Sub-samples were taken from the archived specimens, scraped free of any debris or encrusting organisms and bleached to remove organic material. Each was ground to fine powder along with 0.1 g NaCl (as a calibration standard), then examined using X-ray diffractometry (XRD) for carbonate mineralogy using the methods described by Smith et al. (1998) and calibration equations from Gray and Smith (2004). Precision of this method is approximately 0.1 wt.% MgCO3 and 1% calcite (Smith et al., 2006). Mineralogical results were tested for significant differences among taxa/clades using an unpaired two-sided t-test (Welch’s t-test) because there were unequal sample sizes and unequal variances in the groups. To test for phylogenetic signal in our mineralogical data we calculated Blomberg’s K (Blomberg and Garland, 2002; Blomberg et al., 2003) using the R (R Development Team, 2010) package picante (Kembel et al., 2010). For those data which have substantial phylogenetic signal, the effect of other variables was examined using Phylogenetic Generalised Least Squares models (PGLS) implemented in the R package caper (Orme et al., 2012). PGLS allows k, a scaling factor that takes into account the degree to which closely related samples are similar due to their shared evolutionary history, to be included in the statistical model. In caper, we fitted k using maximum likelihood and compared the bestfit model to models with k = 0 (no phylogenetic signal) and k = 1 (all variance in the data explained by the phylogenetic tree). By using the maximum likelihood estimate of k where appropriate we were able to co-estimate the degree to which phylogenetic signal should be controlled in our analyses and our parameters of interest given that statistical correction.

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Acknowledgements We thank Damian Walls, University of Otago for XRD support. We gratefully acknowledge permission granted by Museum of New Zealand Te Papa Tongarewa to sample coralline algal collections, and the assistance given by Te Papa’s Botany staff, particularly Jenn Dalen. We acknowledge the funding provided by Ministry of Fisheries Biodiversity Fund (ZBD200105, ZBD200407) for initial collection of the majority of specimens from Te Papa’s collection that were sampled for this study. We thank Dr. Wendy Nelson, National Institute of Water and Atmospheric Research (NIWA), Prof. Hamish Spencer, Allan Wilson Centre for Molecular Ecology and Evolution, Department of Zoology, University of Otago, and two anonymous reviewers for helpful comments during the preparation of this paper. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytochem.2012. 06.003. References Adey, W.H., MacIntyre, I.G., 1973. Crustose coralline algae: A re-evaluation in the geological sciences. Geol. Soc. Am. Bull. 84, 883–904. Andersson, A.J., Mackenzie, F.T., Bates, N.R., 2008. Life on the margin: Implications of ocean acidification on Mg-calcite, high latitude and cold-water marine calcifiers. Mar. Ecol. Prog. Ser. 373, 265–273. Bass-Becking, L.G., Galliher, E.W., 1931. Wall structure and mineralization in coralline algae. J. Phys. Chem. 35, 467–479. Blomberg, S.P., Garland Jr., T., Ives, A.R., 2003. Testing for phylogenetic signal in comparative data: Behavioral traits are more labile. Evolution 57, 717– 745. Blomberg, S.P., Garland Jr., T., 2002. Tempo and mode in evolution: Phylogenetic inertia, adaptation and comparative methods. J. Evol. Biol. 15, 899–910. Borowitzka, M.A., Larkum, A.W.D., Nockolds, C.E., 1974. A scanning electron microscope study of the structure and organization of the calcium carbonate deposits of algae. Phycologia 13, 195–203. Bosence, D.W.J., 1991. Coralline algae: Mineralogy, taxonomy, and palaeoecology. In: Riding, R. (Ed.), Calcareous Algae and Stromatolites. Springer-Verlag, Berlin, pp. 98–113. 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. Phylogenet. Evol. 46, 958–973. Chave, K.E., 1954. Aspects of the biogeochemistry of magnesium. I. Calcareous marine organisms. J. Geol. 62, 266–283. Chisholm, J.R.M., 2000. Calcification by crustose coralline algae on the northern Great Barrier Reef, Australia. Limnol. Oceanogr. 45, 1476–1484. Clark, D., Lamare, M., Barker, M., 2009. Response of sea urchin pluteus larvae (Echinodermata: Echionidea) to reduced seawater pH: A comparison among a tropical, temperate, and a polar species. Mar. Biol. 156, 1125–1137. Clarke, F.W., Wheeler, W.C., 1922. The inorganic constituents of marine invertebrates. US Geol. Surv. Prof. Pap. 124, 62. Doney, S.C., Fabry, V.J., Feely, R.A., Kleypas, J.A., 2009. Ocean acidification: The other CO2 problem. Ann. Rev. Mar. Sci 1, 169–192. Farr, T., Broom, J., Hart, D., Neill, K., Nelson, W., 2009. Common coralline algae of northern New Zealand: An identification guide. NIWA Information Series No. 70. Available from: . Fine, M., Tchernov, D., 2007. Scleractinian coral species survive and recover from decalcification. Science 315, 1811. Flajs, G., 1977. Die ultrastrukturen des kalkalgenskeletts (Skeletal ultrastructures of calcareous algae). Palaeontogr. Abt. B 160, 69–128. Felsenstein, J., 1985. Phylogenies and the comparative method. Am. Nat. 125, 1–15. Fragoso, D., Ramírez-Cahero, F., Rodríguez-Galván, A., Hernández-Reyes, R., Heredia, A., Rodríguez, D., Aguilar-Franco, M., Bucio, L., Basiuk, V.A., 2010. Characterization of the CaCO3 biomineral in coralline red algae (Corallinales) from the Pacific coast of Mexico. Cienc. Mar. 36, 41–58. Gamboa, G., Halfar, J., Hetzinger, S., Adey, W., Zack, T., Kunz, B., Jacob, D.E., 2010. Mg/Ca ratios in coralline algae record northwest Atlantic temperature variations and North Atlantic Oscillation relationships. J. Geophys. Res. 115, C12044. http://dx.doi.org/10.1029/2010JC006262. Gao, K., Aruga, Y., Asada, K., Ishihara, T., Akano, T., Kiyohara, M., 1993. Calcification in the articulated coralline alga Corallina pilulifera, with special reference to the effect of elevated CO2 concentration. Mar. Biol. 117, 129–132.

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