MULTI-GENE PHYLOGENETIC ANALYSES OF NEW ZEALAND CORALLINE ALGAE:CORALLINAPETRA NOVAEZELANDIAE GEN. ET SP. NOV. AND RECOGNITION OF THE

HAPALIDIALES ORD. NOV.1

Wendy A. Nelson2

National Institute of Water and Atmospheric Research, Private Bag 14-901, Wellington 6241, New Zealand

School of Biological Sciences, University of Auckland, Private Bag 92-019, Auckland 1142, New Zealand

Judith E. Sutherland

School of Biological Sciences, University of Auckland, Private Bag 92-019, Auckland 1142, New Zealand

Tracy J. Farr

Royal Society of New Zealand, PO Box 598, Wellington 6140, New Zealand

Darren R. Hart

Department of Biochemistry, University of Otago, PO Box 56, Dunedin 9054, New Zealand

Kate F. Neill

National Institute of Water and Atmospheric Research, Private Bag 14-901, Wellington 6241, New Zealand

Hee Jeong Kim, and Hwan Su Yoon

Department of Biological Sciences, Sungkyunkwan University, Suwon 440-746, Korea

Coralline red algae from the New Zealand regionwere investigated in a study focused on documentingregional diversity. We present a multi-gene analysisusing sequence data obtained for four genes (nSSU,psaA, psbA, rbcL) from 68 samples. The studyrevealed cryptic diversity at both genus and specieslevels, confirming and providing further evidence ofproblems with current taxonomic concepts in theCorallinophycidae. In addition, a new genusCorallinapetra novaezelandiae gen. et sp. nov. iserected for material from northern New Zealand.Corallinapetra is excluded from all currentlyrecognized families and orders within theCorallinophycidae and thus represents a previouslyunrecognized lineage within this subclass. We discussrank in the Corallinophycidae and propose the orderHapalidiales.

Key index words: Corallinales; Corallinapetra novaeze-landiae gen. et sp. nov.; Hapalidiales ord. nov.;multi-gene phylogeny; New Zealand; nSSU; psaA;psbA; rbcL

Abbreviations: nSSU, nuclear small subunit rRNAgene; OTU, operational taxonomic unit; psaA, pho-tosystem I P700 chlorophyll a apoprotein A1 gene;psbA, photosystem II P680 reaction center D1 pro-

tein gene; rbcL, ribulose-1,5-bisphosphate carboxyl-ase/oxygenase large subunit gene

Calcified marine red algae have been receivingincreasing levels of attention over the past decade.Coralline algae occupy habitats from intertidalshores to deep subtidal regions, from the tropics tothe poles (Nelson 2009). Both geniculate and non-geniculate taxa have been reported to harbor a richdiversity of associated organisms, playing criticalfunctional and structural roles in coastal ecosystems(e.g., Barber�a et al. 2003, Kamenos et al. 2004a,b,Foster et al. 2007, Pe~na and B�arbara 2008a,b, Cow-les et al. 2009, Berlandi et al. 2012) includinginvolvement in the metamorphosis of larval stagesof key invertebrate taxa (Roberts 2001, Harringtonet al. 2004). As calcified organisms, they are vulnera-ble to the impacts of anthropogenic climate change,particularly ocean acidification (e.g., Martin andGattuso 2009, Basso 2012, Cornwall et al. 2013a,b,Martin et al. 2013, McCoy 2013).Over the past decade, there has been renewed

vigor in taxonomic studies of coralline algae. Recentresearch employing DNA sequence data and phylo-genetic analyses is enabling a fresh evaluation ofthese algae and their relationships. Traditionally,corallines have been regarded as difficult to study.Detailed anatomical investigations of reproductivestructures in calcified organisms are both time con-suming and technically challenging, and this is cou-

1Received 5 October 2014. Accepted 11 December 2014.2Author for correspondence: e-mail wendy.nelson@niwa.co.nz.Editorial Responsibility: P. Gabrielson (Associate Editor)

J. Phycol. *, ***–*** (2015)© 2015 Phycological Society of AmericaDOI: 10.1111/jpy.12288

1

pled with the recognition that the application ofnames to species and genera “has been based ontradition rather than being firmly linked to typespecimens” (Woelkerling 1988). Non-geniculate cor-alline algae have also been regarded as highly mor-phologically variable (Woelkerling et al. 1993), withthe understanding that a single species may exhibita variety of growth forms, making recognition in thefield by morphological features alone difficult, ifnot impossible (Woelkerling 1996a: 196, 206, b:230). The use of DNA sequence data to assist withidentification has greatly aided taxonomic investiga-tions, and, critically, it has enabled the reliability oftraditional taxonomic characters to be re-evaluated.

The order Corallinales was formally established bySilva and Johansen (1986) and members are charac-terized by the presence of calcium carbonate intheir cell walls, and the possession of pit plugs withtwo cap layers. Le Gall and Saunders (2007) pro-posed the subclass Corallinophycidae within theFlorideophyceae, based on a nuclear DNA sequencephylogeny, initially comprised of the orders Coralli-nales and Rhodogorgonales (Fredericq and Norris1995). More recently, the order Sporolithales wassegregated from Corallinales to recognize the dis-tinctiveness of the members of the family Sporolith-aceae (Le Gall et al. 2010).

The recognition of the group at subclass level issupported by the age of the group: coralline algaehave comparatively well documented fossil recordsincluding the 600 million years old (Mya) Neoprote-rozoic Doushantuo (China) algal fossil that isassigned to the ancestral group of corallines (Xiaoet al. 1998, 2004). Biocalcified corallines were foundin Paleozoic sediments (Johnson 1956, Brooke andRiding 1998), followed by diversification of moderncorallines during the Cretaceous and Tertiary peri-ods (Aguirre et al. 2000).

Woelkerling and Nelson (2004) provided a base-line summary and analysis of the taxonomic biodi-versity of coralline red algae reported from the NewZealand region: although more than 80 species andinfraspecific taxa have been recorded, many of theserecords need to be verified and re-evaluated in amodern context (i.e. combining morphology, andanatomy with molecular data). Although the firstpublished record of coralline algae from the NewZealand region was reported by Lamouroux (1821),there have been no monographic treatments of thecoralline flora in New Zealand. Two major studies(Harvey et al. 2005, Farr et al. 2009) have been con-ducted in the past decade focused on collecting rep-resentative samples from a wide range of habitats,identifying a DNA-based marker for screening sam-ples, and then applying this to provide an overviewof genetic diversity present in the region. Broomet al. (2008) identified the utility of psbA for DNA-based screening of diversity in coralline algae, wid-ening the range of identifiable specimens to includesterile and abraded material. This work was accom-

panied by morphological and anatomical investiga-tions to characterize taxa, linking these combineddata sets to generic and species concepts. Twoguides to commonly occurring species in centraland northern New Zealand were prepared (Harveyet al. 2005, Farr et al. 2009), and a large database ofsequence data was assembled to inform furtherresearch.Over the past 5 years relationships of calcified red

algae have been investigated employing analyseswith a range of genetic markers (e.g., Broom et al.2008, Le Gall et al. 2010, Bittner et al. 2011, Katoet al. 2011). These have enhanced clarity in somegroups, and revealed problems with generic andspecific concepts in other groups. In addition, therehave been studies specifically targeting certain gen-era, some of which employed both morphologicaland DNA sequence data, while others primarilyfocused on one or other of these sources of data(e.g., Harvey et al. 2009a,b, Walker et al. 2009, Ga-brielson et al. 2011, Martone et al. 2012, Hind andSaunders 2013, Kato et al. 2013).This paper documents the phylogenetic diversity of

coralline algae in the New Zealand region, usingDNA sequence data to provide an overview and con-text for subsequent taxonomic investigations as inearlier studies (e.g., Bangiales: Broom et al. 2004,Nelson et al. 2006, Sutherland et al. 2011; Ulvaceae:Heesch et al. 2009; Kallymeniaceae: D’Archino et al.2011). We have utilized a two step approach, usingpsbA sequence data to identify well supported clades,followed by a multi-gene phylogenetic analysis of rep-resentatives of these clades using a further threemarkers, two plastid (psaA and rbcL) and one nuclear(nSSU). We describe a new genus from New Zealandthat does not conform to any existing family conceptsin the Corallinophycidae and propose the elevationof the family Hapalidiaceae to ordinal level, and pro-vide an emended concept of the Corallinales. Surveysof sequence diversity and phylogenetic analyses suchas this study have provided insights that have applica-tion beyond the confines of the regional flora. Thedata presented here will help to inform futureresearch programs within Australasia, but also illumi-nate particular issues that have relevance worldwide,particularly with respect to the examination of taxo-nomic concepts at species level and above.

MATERIALS AND METHODS

Collections and taxon sampling. Non-geniculate and genicu-late coralline algal samples were collected from locationsaround New Zealand, with collections focused particularly oncentral and northern New Zealand as part of two surveys con-ducted from 2001 to 2008. The geographic range of collec-tions extends from the Kermadec Islands in the north (29°S) to Stewart Island in the south (51° S), and from the westcoast of the South Island (~163° E) to the Chatham Islands(176° W) east of mainland New Zealand. Table S1 in the Sup-porting Information gives collection information for speci-mens used in this study.

2 WENDY A. NELSON ET AL.

Specimens were inspected and cataloged, and a portionwas subsampled and dried rapidly in desiccant silica gelwithin a few hours of collection where possible for latermolecular analysis. Species identifications are based on Har-vey et al. (2005) and Farr et al. (2009) for all New Zealandcoralline taxa. Vouchers of all New Zealand specimens usedin the analyses were deposited at WELT (Herbarium,Museum of New Zealand Te Papa Tongarewa, Wellington;Thiers 2014).

Coralline algal identification. Coralline specimens were pre-pared for identification following methods described in previ-ous studies (e.g., Harvey et al. 2005, Harvey and Bird 2008).Small samples of fertile material were decalcified in diluteHNO3, stained with 5% aqueous KMnO4, dehydrated throughchanges to 100% ethanol, then embedded in “LR White”resin (London Resin Co., Reading, Berkshire, UK). Perma-nent slides were prepared: sections 10 lm thick were cut witha slide microtome, cleared with HistoClear (National Diag-nostics, Atlanta, GA, USA), and mounted with Eukitt (Sigma-Aldrich, Auckland, New Zealand). These slides were depos-ited with voucher specimens held at WELT. Identificationswere based on comparisons of vegetative and reproductivefeatures with previously published data from New Zealand(Harvey et al. 2005, Farr et al. 2009), Australia (Womersley1996, Harvey et al. 2003b, 2006) and elsewhere.

DNA extractions, PCR amplification and sequencing. DNA wasextracted using a Qiagen Tissue DNA Extraction Kit (Qiagen,Hilden, Germany) using a modified protocol as in Broomet al. (2008). The psbA gene was amplified and sequencedusing primers psbAF1 and psbAR2 (Yoon et al. 2002). ThenSSU gene was either amplified in a single reaction usingprimers G01/G04 (Saunders and Kraft 1994), or 18e/J04(Hillis and Dixon 1991, Broom et al. 1999), or as two overlap-ping pieces using primers 18e and J05 (Broom et al. 1999),and G04/J04 and sequenced using the PCR primers and com-binations of internal primers G10, G02 and G06 (Saundersand Kraft 1994) as necessary. The rbcL and psaA genes wereamplified and sequenced with primers rbcL090F/R-rbcS start(Freshwater and Rueness 1994, Yoon et al. 2002) andpsaA130F/psaA1600R or psaA1760R (Yoon et al. 2002),respectively. PCR products were purified and sequencedusing standard methods.

Sequence and phylogenetic analyses. Partial psbA sequences upto 850 bp in length were obtained for 533 coralline speci-mens. Sequences were aligned using Geneious V6.1.6 (Bio-Matters, Auckland, New Zealand), and a maximum likelihood(ML) tree was estimated using PhyML V3.0 (Guindon et al.2010), with support assessed by the approximate likelihoodratio test (Anisimova and Gascuel 2006). The tree wasinspected for well-supported terminal clades, and a represen-tative specimen from each clade was selected for furthersequencing of the nSSU, rbcL and psaA genes. Our criteriafor selection were that the sequences were representative of amonophyletic group with little sequence variation, or thatthey were singleton taxa, varying by more than 12 substitu-tions from sequences in their sister clades. Thus our criteriacombined both tree-based and distance-based assessment ofoperational taxonomic unit (OTU) boundaries (Meier et al.2006). The selected specimens included representatives ofcommon taxa identified in central and northern New Zea-land (Harvey et al. 2005, Farr et al. 2009), as well as speci-mens for which generic or species placement was unclear. Wealso included members of genus Corallina which showed onlysmall variation in psbA sequence but which varied in mor-phology, and two sequences from specimens assigned to Meso-phyllum erubescens (Foslie) Me. Lemoine which differed byonly 3 substitutions (ND171 and ND318). These 64 selectedspecimens constituted the dataset analyzed in this article.

Sequences from each of the four markers were alignedusing Geneious and concatenated using SequenceMatrix (Va-idya et al. 2011). Unalignable regions of the nSSU gene wereremoved before analysis. Phylogenetic datasets were con-structed for each marker individually and for all markers con-catenated. We included as outgroup taxa sequences fromGracilaria secundata Harvey, Ceramium kondoi Yendo, Audouinel-la sinensis C. C. Jao, Thorea violacea Bory, Palmaria palmata (L.)F. Weber & D. Mohr, and Nemalion sp. N10. These taxa werechosen because sequences for at least three of our loci wereavailable from the same individual, minimizing the possibilityof chimeric sequences in our multi-gene dataset. We alsoincluded sequences from two representatives of the Rhodo-gorgonales, Renouxia sp. and Rhodogorgon carriebowensis J. N.Norris & Bucher, as well as two taxa from Australia and onefrom Japan (Amphiroa sp. Aus, Sporolithon durum (Foslie) R. A.Townsend & Woelkerling and Neogoniolithon brassica-florida(Harvey) Setchell & L. R. Mason), which are congeneric withNew Zealand taxa, and two geniculate corallines, Corallinapilulifera Postels & Ruprecht and Calliarthron tuberculosum(Postels & Ruprecht.) E. Y. Dawson, for which sequence dataare available for our markers. Each OTU in the analysis rep-resents sequence data from 2–4 markers from the same speci-men with the exception of Corallina officinalis L. NZC2537/ASE091, Arthrocardia sp. NZC2598/NZC2021, Sporolithondurum NZC2375/NZC2312 and Heydrichia homalopasta R. A.Townsend & Borowitzka NZC2015/NZC0748. For these weconcatenated sequences from two separate specimens fromthe same psbA clade in order to maximize available sequencedata. We also concatenated the nSSU sequence from Renouxiasp. HV508 (EF033584.1) and the rbcL sequence from Reno-uxia antillana Fredericq & J.N. Norris (U04181.1), the onlysequences available for Renouxia, to represent that genus, andthe nSSU sequence from a specimen of Calliarthron tuberculo-sum (U60944.1) with three sequences derived from a com-plete plastid sequence of a separate specimen (KC153978.1)to represent that taxon. GenBank accession numbers for taxain the phylogenetic analyses are given in Table S2 in the Sup-porting Information.

Appropriate models of sequence evolution were assessedfor each dataset using jModeltest 2 (Darriba et al. 2012) forthe nSSU dataset and PartitionFinder V1.1.1 (Lanfear et al.2012) for datasets consisting of the three coding regions andfor the four-gene concatenated dataset. PartitionFinder alsoestimated an appropriate partitioning strategy for the psaA,psbA and rbcL datasets and for the concatenated dataset.These strategies were used in subsequent MrBayes analyses,and are given in Table 1. RAxML analyses used the GTR+Γ

TABLE 1. Partitioning strategies and models of sequenceevolution used for the 4-gene concatenated dataset. Parti-tions 1–7 are for the DNA dataset, partitions 8, 9 and 10are the partitions for the amino acid portion of the mixedDNA and protein dataset.

Partition Model of sequence evolution

1: nSSU Kimura80+I+Γ2: psaA codon 1+ rbcL codon 1 SYM+I+Γ3: psbA codon 1 SYM+I+Γ4: psaA codon 2+ psbAcodon 2+ rbcL codon 2

GTR+I+Γ

5: psaA codon 3 GTR+I+Γ6: psbA codon 3 GTR+I+Γ7: rbcL codon 3 GTR+I+Γ8: psaA amino acid sequence CpREV9: psbA amino acid sequence MtArt10: rbcL amino acid sequence LG

MULTI-GENE ANALYSIS OF NZ CORALLINE ALGAE 3

model of sequence evolution, because that program offersfewer options for choice of models.

Trees for individual genes and for the 4-gene concatenateddataset were assessed under the ML criterion using the rapidbootstrap option (1,000 replicates) in RAxML (Stamatakis2006, Stamatakis et al. 2008). The data were partitionedaccording to the optimal schemes estimated by PartitionFinder.We also ran the 4-gene analysis without the two Neogoniolithontaxa, which are resolved on long branches, to investigate theimpact of these long branch taxa on resolution in the tree.

Bootstrapped ML trees were also estimated using the sameprocess for two amino acid datasets to test whether these gavea topology consistent with the DNA data. We translated thethree coding gene sequences using SeaView 4.4.2 (Gouy et al.2010) and used PartitionFinder to estimate appropriate parti-tions and models of sequence evolution. We then estimatedML bootstrapped trees using the rapid bootstrap option inpRAxML (Stamatakis 2006, Stamatakis et al. 2008), under 100bootstrap replicates for a dataset consisting of the threeamino acid sequences concatenated, and for a second datasetconsisting of the three amino acid sequences concatenatedwith the nSSU DNA data. Each marker was defined as a sepa-rate partition; models of sequence evolution used are givenin Table 1.

Bayesian analysis of the 4-gene concatenated DNA data setwas performed using MrBayes V3.2.1. The partitioningscheme and associated models of sequence evolution werethose identified by PartitionFinder V1.1.1 (Table 1), exceptthat we chose to use 6 gamma rate categories, rather than 4gamma rate categories plus an invariant sites parameter.Branch length prior means were set to 0.01. Parameters wereallowed to vary among partitions, and the Markov chainMonte Carlo was run for 8,000,000 generations. We ran twoseparate analyses of 4 chains each. Burnin was assessed usingTracer V1.5 (http://tree.bio.ed.ac.uk/software/tracer/) byinspection of log-likelihood plots and average parameter val-ues, and was confirmed by inspection of PSRF scores calcu-lated by the sump function of MrBayes.

Sequences from collection NZC2381 were compared tosequences in GenBank using BLAST (Altschul et al. 1990),implemented as Megablast at http://www.ncbi.nlm.nih.gov/blast/Blast.cgi (last accessed 17 July 2014).

RESULTS

Numbers of sequences in each of the phyloge-netic matrices are given in Table 2. In our concate-nated dataset 46 taxa were represented by fourmarkers, 21 by three, and seven by two sequencesonly. Taxa included in the phylogenetic datasetwere identified on the basis of published morpho-anatomical criteria, and on identifications made bythe authors of sequences obtained from GenBank;

sequencing of type material to confirm identifica-tions was outside the scope of this study.Taxa in the phylogenetic dataset showed consider-

able variation among psbA DNA sequences: mem-bers of the Hapalidiaceae and Corallinaceae wereseparated by from 64 substitutions (Mesophyllummacroblastum (Foslie) W.H. Adey NZC2369 – Spong-ites sp. NZC0686) to 152 substitutions (Neogoniolithonsp. NZC2043 – Mesophyllum engelhartii (Foslie) W.H.Adey NZC2236). Members of Corallina were sepa-rated by 4–14 substitutions. It is notable that thepsbA sequence for Corallina pilulifera from Japan dif-fered from that of the common and readily identifi-able New Zealand taxon, referred to here asCorallina officinalis, by only six substitutions,although it appears to be morphologically distinct,supporting the hypothesis that interspecific psbAsequence variation within Corallina as currentlyunderstood is small.Analyses of individual genes showed no significant

incongruencies among the trees (data not shown).The removal of the two long-branch taxa (Neogonioli-thon brassica-florida Ryuku and Neogoniolithon sp. NZ)had no impact on the topology of the concatenatedtree, but did result in increased support for mono-phyly of the clade consisting of Arthrocardia, Calliar-thron and Corallina (from 79% to 100%) and forthe Corallinoideae overall (Jania, Arthrocardia, Cal-liarthron and Corallina, from 65% to 100%). Onlythe results of the concatenated analysis includingNeogoniolithon are shown here (Fig. 1). The concate-nated nSSU and amino acid data set, and the three-gene amino acid data set generated trees that weregenerally consistent in topology with the DNA analy-ses, but with much lower resolution. Bootstrap sup-port (BS) values from the concatenated nSSU andamino acid analysis are shown in Figure 1, but thediscussion of support in the text is restricted to theresults of the DNA analyses.In the concatenated 4-gene DNA analysis, families

Sporolithaceae, Rhodogorgonaceae, Hapalidiaceaeand Corallinaceae, were each supported as mono-phyletic (Fig. 1). Support for the monophyly of theRhodogorgonaceae was very strong with ML BS of100% and Bayesian posterior probability of 1. Mono-phyly of the Corallinaceae was also supported at100/1. Support for monophyly of the remaining twoextant families was lower but still significant, withmonophyly of the Hapalidiaceae supported at 92/1,and monophyly of the Sporolithaceae at 71/1. A sis-ter relationship between the Rhodogorgonaceaeand Sporolithaceae was not recovered, but a sisterrelationship between the Corallinaceae and Hapalid-iaceae was recovered with support 100/1.Within Sporolithaceae, Heydrichia was represented

by two specimens that were identified to speciesbased on morphological characters. These OTUswere not resolved as being monophyletic. The analy-ses point to the presence of two distinct species ofSporolithon in New Zealand. Epilithic and rhodolith-

TABLE 2. Numbers of ingroup and outgroup taxa repre-sented in each of the phylogenetic matrices. Rhodogorgo-nales sequences are counted as part of the ingroup.

DatasetNumber of

outgroup taxaNumber ofingroup taxa

psaA 5 57psbA 6 67rbcL 6 53nSSU 4 634-gene data set 6 68

4 WENDY A. NELSON ET AL.

forming specimens from New Zealand that havebeen identified as Sporolithon were found to beclearly distinct from each other. The rhodolith-

forming Sporolithon specimens from New Zealandwere shown to be more closely related to thesequence data from Australian specimens identified

0.2

Corallina pilulifera

Corallina sp. NZC2113

Heydrichia homalopasta NZC2015 / NZC0748

Sporolithon durum rhodolith NZC2375 / NZC2312

Lithophyllum johansenii NZC2055

Arthrocardia sp. NZC2598 / NZC2021

Mesophyllum erubescens NZC2051

Corallina officinalis NZC2537 / ASE091

Jania rosea NZC2022

Neogoniolithon brassica-florida RyukuHydrolithon improcerum NZC0667

Spongites sp. NZC2125

Lithophyllum carpophylli NZC2115

Mesophyllum erubescens NZC0476

Lithophyllum riosmenae NZC2487

Mesophyllum erubescens NZC0709

Amphiroa sp ASZ198 Aus

Nemalion sp. N10

Hapalidiaceae NZC2090

Thorea violacea

Synarthrophyton patena DH20 Aus

Spongites sp. NZC0090

Lithophyllum corallinae NZC2250

Corallina sp. ASD200

Spongites sp. NZC0686

Heydrichia woelkerlingii NZC2014

Calliarthron tuberculosum

Hapalidiaceae NZC0747

Palmaria palmata

Mesophyllum printzianum NZC2342Mesophyllum erubescens NZC2365

Phymatolithon repandum NZC2501

Corallina sp. ASD026

Mesophyllum erubescens NZC2433

Jania sp. ASE281

Mesophyllum engelhartii NZC2236

Jania rosea NZC2554

Amphiroa anceps NZC2361 / NZC2301

Audouinella sinensis

Mesophyllum erubescens NZC2162

Mesophyllum macroblastum NZC2288

Lithophyllum sp. NZC0314

Corallina sp. GS08

Sporolithon durum Aus

Corallinapetra novaezelandiae NZC2381

Lithothamnion crispatum NZC2315

Arthrocardia sp. ASE107

Pneophyllum fragile NZC2019

Rhodogorgon carriebowensisRenouxia sp.

Jania verrucosa ASD196

Gracilaria secundata

Synarthrophyton patena NZC0899

Neogoniolithon sp. NZC2043

Lithophyllum stictaeforme NZC2130

Jania sagittata NZC2389

Spongites sp. NZC0777

Corallina sp. NZC2293

Arthrocardia sp. NZC2539

Lithophyllum pustulatum NZC2505

Sporolithon sp. epilithic NZC2175

Spongites sp. NZC2009

Synarthrophyton schielianum ASE297

Porolithon onkodes NZC2547

Spongites sp. NZC2122

Jania sp. NZC2234

Mastophora pacifica NZC2000

Corallina sp. NZC2496

Phymatolithon sp. NZC2013

Mesophyllum macroblastum NZC2369

Pneophyllum coronatum NZC0730

Ceramium kondoi

Hapalidiaceae MATS 2166

Lithophyllum sp. NZC2545

93/1

70/0.98

71/1

97/1

97/1

85/1

98/1

94/1

79/1

94/0.93

97/1

85/1

61/1

97/1

58/1

99/1

70/0.98

69/0.77

85/0.63

94/1

68/0.94

98/1

98/1

92/1

65/1

82/1

99/1

*

68/-

98/1

-/0.99

** *

**

**

*

*100/1

*

*

*

*

**

*

*

** *

100

88

59

-

39

37

29

70 97-

36

100/150

100/1

68

9770

100/1100/1

-

-

-100/1

-

54

82

8387

62

-

40

98

100/175

-

-

92

-

-74

- 76 79 -

100/1

9345

100/1100/1

-

100/1

-

Sporolithaceae, Sporolithales

Rhodogorgonaceae, Rhodogorgonales

Hapalidiaceae, Hapalidiales

Corallinaceae, Corallinales

FIG. 1. ML phylogeny of the concatenated data set estimated by RAxML. ML Bootstrap support values (1,000 replicates) and posterior prob-abilities (PP) estimated by MrBayes for the DNA data set are shown to the left of each node above the line, and bootstrap support values (100replicates) from the amino acid level analysis are shown below. Only bootstrap values greater than 65 and PP values greater than 0.8 are shown.Where one support value reaches the cut-off value, all are shown. Nodes that received 100/1/100 support are indicated with an asterisk.

MULTI-GENE ANALYSIS OF NZ CORALLINE ALGAE 5

as Sporolithon durum than to epilithic New Zealandspecimens.

Notably, the collection NZC2381 from northeast-ern New Zealand was resolved as a sister taxon tothe combined Hapalidiaceae and Corallinaceaeclade. The 100/1 support for this relationship alsoexcludes it from the Sporolithales and Rhodogorgo-nales. This taxon is therefore excluded from all cur-rently recognized families in the Corallinophycidae.BLAST analysis showed that the nSSU sequence ofNZC2381 was 99% homologous to an unidentifiedCorallinaceae specimen collected at Beachport,South Australia in 2003 (GenBank accessionAY247408.1, differing by 7 substitutions and oneindel). A new genus and species is described belowto accommodate the northern New Zealand taxon.

The Hapalidiaceae was resolved as monophyleticand a number of well supported clades wereresolved within it. Synarthrophyton was well supportedas a genus, consisting of three taxa each on rela-tively long branches. Bailey and Chapman (1998)and Bailey et al. (2004) included sequence datafrom a South Australian specimen identified as Syn-arthrophyton patena (Hooker & Harvey) R.A.Town-send in their nSSU analyses (GenBank accession no.U61255.1). The nSSU sequence of our Australianmaterial differs from Bailey’s by two substitutionsand three single base pair indels, and from thatrecently obtained by Scott et al. from Victorianmaterial (KC157578.1, published only in GenBank)by one substitution and three single base pair in-dels. Numbers of substitutions between sequencesof our New Zealand and Australian specimens are:nSSU, 19/945; psaA, 132/1299; psbA, 55/804. Thereare no rbcL data for S. patena DH20 Aus.

Mesophyllum was not resolved as monophyletic.The assignment of species identifications based onanatomical and morphological characters is clearlyproblematic as there were two specimens identifiedas M. macroblastum, one as M. engelhartii, one asM. printzianum Woelkerling & A.Harvey, and six asM. erubescens, and long branches separate these spec-imens. Phymatolithon was also not resolved as mono-phyletic in the analyses, with P. repandum (Foslie)Wilks & Woelkerling only distantly related to thealga identified as Phymatolithon sp. There was only asingle species of Lithothamnion represented in thisstudy, the rhodolith-forming L. crispatum Hauck.There were three OTUs within the family Hapalidia-ceae that were not assigned to a genus based onanatomical or morphological characters (NZC2090,NZC0747 and MATS 2166).

Within Corallinaceae three genera of geniculatecoralline algae (Jania, Arthrocardia and Corallina)were each resolved as monophyletic with strong sup-port, and contained unnamed or unidentified taxa.There were six species of Jania differentiated inthese analyses and three species of Arthrocardia.Seven specimens of Corallina from New Zealandwere resolved in a clade with Corallina pilulifera,

showing some sequence variation but with littleinternal resolution in the analyses presented here.A single species from New Zealand has been

placed in Neogoniolithon based on morphology andsequence data, although it was not closely related toany sequences of this genus currently in GenBank.The material identified as Mastophora pacifica (Hey-drich) Foslie was distinct from all other New Zea-land members of the family and is the onlyrepresentative of the sub-family Mastophoroideaeknown in the New Zealand flora to date (Kato et al.2011).Two specimens identified as Hydrolithon improce-

rum (Foslie & M. Howe) Foslie and Porolithon onkodes(Heydrich) Foslie were not resolved as monophy-letic in our analysis. The analyses resolved two well-supported clades that include six specimens thathad been identified as Spongites along with two spec-imens of Pneophyllum.A monophyletic group consisting of Lithophyllum

and Amphiroa was well supported in the analyses,but a non-geniculate species identified as Lithophyl-lum riosmenae A.Harvey & Woelkerling was resolvedas a sister to Amphiroa, clearly distinct from theother members of the genus Lithophyllum. Amphiroawas a well supported genus with a single New Zea-land specimen, which has been known as A. anceps(Lamarck) Decaisne Excluding L. riosmenae, thegenus Lithophyllum was represented in these analysesby seven specimens, five of which have beenassigned names.Taxonomic results. Corallinapetra T. J. Farr, W. A.

Nelson & J.E. Sutherland gen. nov.Figures 2–4Non-geniculate, crustose, smooth to slightly tex-

tured surface; flared epithallial cells, cell fusions;gametophytic phase with uniporate conceptacles;sporophytic phase with individual compartmentsgrouped in shallow depressions; small pores visibleon thallus surface giving appearance of multiporateconceptacles; stalk cells.Type species: Corallinapetra novaezelandiae sp. nov. T.

J. Farr, W. A. Nelson & J.E. SutherlandEtymology: Named for the calcified growth on

stonesComments: This genus is based on a single collec-

tion, recognized initially on the basis of sequencedata and consequent phylogenetic placement, andfurther characterized by anatomical and morpholog-ical observations.Corallinapetra novaezelandiae T. J. Farr, W. A. Nel-

son & J.E. Sutherland sp. nov.With the characters of the genus.Holotype: WELT A032914 (NZC2381), D. Freeman,

7 April 2006 (Fig. 2a), GenBank KM369069,FJ361637.1, KM369124, FJ361374.1Type Locality: New Zealand, northeastern North

Island, Stephenson Island.Etymology: in reference to the region where this

species has been found.

6 WENDY A. NELSON ET AL.

Comments: Corallinapetra novaezelandiae was col-lected on a single occasion from an offshore islandgroup in the northeastern North Island. All material

FIG. 2. Corallinapetra novaezelandiae. (a) Holotype of Corallina-petra novaezelandiae (WELT A032914); scale bar = 1 cm. (b) Cross-section showing flared epithallial cells; scale bar = 20 lm. (c)Cross-section showing cell fusions; scale bar = 20 lm.

FIG. 3. Corallinapetra novaezelandiae. (a) Surface view of thalluswith uniporate conceptacles; scale bar = 2 mm. (b) Cross-sectionof uniporate conceptacle with depressed pore; scale bar = 50 lm.

FIG. 4. Corallinapetra novaezelandiae. (a) Surface view of thallusshowing depressions with very fine pores; scale bar = 2 mm. (b)Cross-section of thallus with three compartments opening to thal-lus surface; scale bar = 20 lm. (c) Cross-section of thallus withfour neighboring compartments and apparent collapse of files ofcells between these; scale bar = 20 lm. (d) Compartment withevidence of basal stalk cell; scale bar = 20 lm.

MULTI-GENE ANALYSIS OF NZ CORALLINE ALGAE 7

(on 5 separate small cobbles) was dried in silica gelat the time of collection. Cellular preservation waspoor, thus interpretation of vegetative anatomy andof reproductive structures has been challenging.

Both flared epithallial cells (Fig. 2b) and cellfusions (Fig. 2c) are present in the tissue. Uniporateconceptacles were present on two of the five cobblescollected (right hand cobbles Fig. 2a) and wereraised with respect to the thallus surface with a slightmoat or depression around the base of the concepta-cle (Fig. 3a). The conceptacle pore was alsodepressed (Fig. 3b). In cross section view there wasevidence of both carposporangia and spermatangia.On three cobbles there were slightly sunken areas ofthe thallus surface with very small pores, giving theappearance of multiporate conceptacles (Fig. 4a). Incross section these were found to be individual com-partments (Fig. 4, b–d). Some compartments were inclose proximity to one another, and the files of cellsbordering the compartments appeared to be collaps-ing, making a larger joint compartment space(Fig. 4c). It is not clear whether this is an artifact ofthe preservation of this tissue or part of the develop-ment of these structures. Despite multiple serial sec-tions no sporangia were found in any compartmentsalthough there was evidence of stalk cells at the baseof some compartments (e.g., Fig. 4d).

Full characterisation of the genus and species,particularly interpretation of the compartmentstructure and sporangial development, awaits fur-ther collections.

DISCUSSION

The discovery of Corallinapetra novaezelandiae fromnortheastern New Zealand points to even greatertaxonomic diversity within the Corallinophycidaethan has been recognized. This genus is closelyrelated to an undescribed taxon collected in south-ern Australia in 2003 (Bailey et al. 2004). Theseauthors suggested on the basis of observations offlared epithallial cells that this taxon “probablybelongs in the genus Sporolithon.” However on thebasis of our multi-gene analyses Corallinapetra isclearly not a member of either Rhodogorgonales, orSporolithales despite the possession of what areassumed to be bi- and or tetrasporangial compart-ments and flared epithallial cells.

Although clearly a member of the Corallinophyci-dae, the familial and ordinal relationships of thisgenus remain unclear. There is full support undereach method of analysis for a sister relationship ofCorallinapetra novaezelandiae with the combined cladeof Hapalidiaceae and Corallinaceae. Thus, thisgenus is excluded from all currently recognizedfamilies in the Corallinophycidae, and consequentlyits ordinal placement is also unclear. We have noterected a family for Corallinapetra, as we considerthis would be premature: further material is neededfor a more detailed developmental and anatomical

study. Comparison of our data with that of Baileyet al. (2004) indicates that either Corallinapetra isnot monotypic or there is a closely related genus insouthern Australia. The characteristics of this south-ern Australian taxon also need to be considered.The results of this study have confirmed the exis-

tence of significant issues in relation to the morpho-logical and anatomical concepts of genera andspecies in current use. In their review of the pub-lished literature on coralline algae in New ZealandWoelkerling and Nelson (2004) noted that although29 of the 80 species and infraspecific taxa recordedhave been described from material collected in NewZealand, their status remains uncertain because thetypes have not been re-examined in a modern con-text. In addition they questioned the application ofnames based on types outside the region and raisedthe issue of undetected taxa because of the rela-tively sparse collection history from the region. Sub-sequent collecting efforts and the application ofboth DNA sequencing and traditional anatomicalinvestigations have provided a great deal of newdata but not resolution of these problems.Diversity in New Zealand: aligning sequence data with

anatomical and morphological concepts. Conflictbetween sequence data and traditional taxonomicconcepts based on morpho-anatomy is immediatelyapparent in the Sporolithaceae for representativesof Heydrichia. Two species have been reported fromNew Zealand based on anatomical and morphologi-cal data: H. homalopasta (type locality: New SouthWales, Australia) and H. woelkerlingii (type locality:South Africa). In our analysis the material identifiedas H. woelkerlingii is not resolved in a monophyleticclade with H. homalopasta, and is sufficiently distinctfrom it to warrant placement of these species in dif-ferent genera, but confirmation of the identity ofHeydrichia species in New Zealand requires clarifica-tion through examination of the type material atgenus and species level.The data presented here indicate that the epilith-

ic and rhodolith forming species of Sporolithon inNew Zealand are distinct from each other at thespecies level, a separation that was not recognizedby Harvey et al. (2005) but which was discussed byFarr et al. (2009). The two New Zealand species arealso distinct from the sequence available from Aus-tralian collections identified as Sporolithon durum.These results suggest that the wide application ofthe name S. durum (e.g., Yoshida 1998, Wynne 2011,Guiry and Guiry 2014, Darrenougue et al. 2013)may not be appropriate, and clarification of the cor-rect application of this name awaits sequence datafrom the type, or material from the type locality(Cape Jaffa, South Australia).Several species in the Hapalidiaceae included in

our analyses could not be placed in genera basedon available morphological and anatomical data.This situation is also seen in the analyses presentedby Bittner et al. (2011) in which several specimens

8 WENDY A. NELSON ET AL.

were represented on distinct branches and labeled“Unidentified Hapalidiaceae.” Both this study andthat of Bittner et al. (2011) demonstrate that sys-tematic and intensive collecting and screening pro-grams focusing on coralline algae are revealingsubstantially greater diversity than previously recog-nized. Based on these results it is highly likely thatnew genera will result from future studies.

The New Zealand rhodolith-forming species ofLithothamnion has been referred to L. crispatum (typelocality: Adriatic Sea). It was first identified in NewZealand as L. indicum Foslie (type locality: Austra-lia), a species that was later synonymized with L. su-perpositum Foslie (type locality: South Africa) byKeats et al. (2000), a species that was then synony-mized with L. crispatum (Basso et al. 2011). Clarifica-tion of the relationships amongst these taxa awaitssequence data from type or topotype material.There are few examples of macroalgae in New Zea-land conspecific with Mediterranean taxa whereasthere are well documented relationships betweenNew Zealand and Australia and to a lesser extentNew Zealand and South Africa (Hommersand2007). Woelkerling and Nelson (2004) documentedseven species described as Lithothamnion based onNew Zealand types for which the generic and spe-cific status remain unconfirmed.

Specimens identified as Phymatolithon in our studyare not resolved as a monophyletic clade. The sepa-ration of material from New Zealand identified asPhymatolithon repandum (type locality: Victoria, Aus-tralia) from other Phymatolithon species is also seenin the analyses of Bittner et al. (2011). In our studywe employed the current anatomical and morpho-logical species concepts for Phymatolithon to assignspecimens (Woelkerling 1996a). Whether speciesoriginally assigned to Leptophytum (a genus with avery checkered nomenclatural history – see Guiryand Guiry 2014) warrant generic separation requiresattention.

Synarthrophyton is well supported as monophyleticin our analyses. The types of both S. schielianum Wo-elk. & M.S. Foster and S. patena (the generitype) arefrom New Zealand locations (Pitt I., Chatham Is.and Flat Pt, Wairarapa, North I., respectively)(Townsend 1979, Woelkerling and Foster 1989).Our results indicate two distinct species have beenidentified as S. patena on the basis of morphologicaland anatomical characters, one based on Australianmaterial and the other on New Zealand material.The sample in our analysis from Australia that hadbeen identified as S. patena (S. patena DH20 Aus,collected from Victoria), although part of the Syn-arthrophyton clade, represents a distinct species basedon the numbers of substitutions present betweenthis and Synarthrophyton patena NZC0899 from NewZealand. This is reflected in the branch lengths inour tree.

The application of morphological and develop-mental criteria when assigning species to the genera

Clathromorphum, Mesophyllum, and Synarthrophyton isnot straightforward, as noted by earlier authors(Woelkerling and Harvey 1992, 1993, Woelkerling1996a, Harvey et al. 2003b, Maneveldt et al. 2007).The data presented here clarify the clade to whichthe generitype of Synarthrophyton and other NewZealand species belong, providing comparative datafor future assignment of species.Our analyses bring no light to the topic of the

relationships of Mesophyllum, but rather emphasizethe problems with generic and specific circumscrip-tion. Although ten specimens were assigned to fourspecies within Mesophyllum on the basis of anatomi-cal features, these entities are more likely to be cor-rectly assigned to nine species in multiple genera.Bittner et al. (2011) found specimens identified asMesophyllum resolved in two distinct clades, one ofwhich they linked to the type species M. lichenoides(J. Ellis) Me. Lemoine. It appears that at least someof the New Zealand species (including M. printzia-num) are in a sister lineage to the clade containingthe type species M. lichenoides. The correct assign-ment of species names in this group awaits resolu-tion of wider questions in the genus.The three genera of geniculate Corallinaceae,

Jania, Arthrocardia and Corallina, although well sup-ported in our analyses, harbor significant taxonomicand nomenclatural issues requiring resolution. Janiacrassa J. V. Lamouroux was described from materialcollected in the fiords of the southwestern South I.of New Zealand, in the same account and followingthe description of J. verrucosa J.V. Lamouroux, basedon material from central America. As J. crassa andmore recently as J. verrucosa, this species has beenreported worldwide, particularly from tropicalregions in the Pacific and Atlantic (e.g., Guiry andGuiry 2014). In our opinion it is most unlikely thata species from the cold waters of Fiordland NewZealand is also found in central America. The cor-rect assignment of species in Jania requires exami-nation of all the names that have been applied inNew Zealand: there are six additional species of Ja-nia and Cheilosporum (now synonymised with Jania,Kim et al. 2007) that have been described based onNew Zealand type specimens, but most of thesenames have dropped from current usage and theconcepts associated with these species are not clear(see Woelkerling and Nelson 2004).In our analyses three species of Arthrocardia are dis-

tinguished. Questions about whether the two namesused in New Zealand (A. corymbosa (Lamarck) Dec-aisne, type locality: South Africa and A. wardii(Harv.) Aresch., type locality: Victoria, Australia) arecorrectly applied to New Zealand material have beenraised for some time (Womersley and Johansen1988, Adams 1994) and remain unresolved.Corallina has received recent attention including

epitypification of the type species, and examinationof diversity in the genus in the northeastern Atlantic(Walker et al. 2009) and north Pacific (Hind and

MULTI-GENE ANALYSIS OF NZ CORALLINE ALGAE 9

Saunders 2013). The New Zealand Corallina officinal-is is resolved as sister taxon to C. pilulifera (typelocality: Siberia) with strong support. New Zealandmaterial examined to date falls into distinct group-ings based on sequence data of the psbA geneincluding the psbA-trnL spacer (Farr et al. 2009),and with some morphological and anatomical fea-tures that support the separation reflected in thesequence data. The name C. officinalis is beingretained for New Zealand material until the statusof these entities is resolved.

The single species from New Zealand that iswithin the Neogoniolithon group requires furtherattention. This warty and intertidal species wasfound in northern New Zealand (Farr et al. 2009),and the sequence data are distinct from that of anyother species in the genus currently in GenBank.

We applied the name Mastophora pacifica (typelocality: Hawaiian Islands) to a single species grow-ing epiphytically on Galaxaura spp., collected fromthe Kermadec Is. (29° S) to the north of the NorthI., which have a subtropical algal flora (Nelson andDalen in press). Sequence data from the type mate-rial or locality of Mastophora pacifica is required toconfirm the identity of this material.

There is a well supported clade with two well sep-arated species currently identified as H. improcerumand P. onkodes. However, the relationship betweenthese specimens and the Spongites/Pneophyllum cladeis only weakly supported. In a review of the Masto-phoroideae Kato et al. (2011) erected several newsub-families including the Porolithoideae (typegenus and species: P. onkodes, formerly Hydrolithononkodes; type locality: Tami Island, Papua New Gui-nea) and the Hydrolithoideae (type genus and spe-cies: Hydrolithon reinboldii (Weber Bosse et Foslie)Foslie; type locality: Muaras Reef, East Kalimantan,Indonesia). Kato et al. (2011) were not able to clar-ify the phylogenetic relationships between the ge-neritype of Porolithon, P. onkodes and three otherspecies of Hydrolithon including H. improcerum (typelocality: Montego Bay, Jamaica), and pointed to theneed for more taxon sampling in future analyses. Itis not known where the New Zealand materialresolves relative to other taxa in these subfamilies.

In this analysis the clade containing specimensidentified as Spongites and Pneophyllum contains eightdistinct taxa. Although two species of Spongites havebeen reported from New Zealand (Nelson 2012), itis not clear how the sequence data we have obtainedrelate to these species. On the basis of anatomicaland morphological features, the name S. yendoi(Foslie) Y.M.Chamb. (type locality: Shimoda, Japan)has been applied to specimens collected from inter-tidal and subtidal habitats in the North, South andChatham Is., and S. tunicata Penrose (type locality:Tasmania) to collections from the central New Zea-land region. Two species of Pneophyllum, P. fragileK€utz. (type locality: Mediterranean Sea) and P. cor-onatum (Rosanoff) Penrose (type locality: Port Phil-

lip Bay, Australia) have been reported from NewZealand and receive moderate support as a mono-phyletic group.Recent accounts of the genus Amphiroa in Australia

(Harvey et al. 2009b, 2013, Woelkerling and Harvey2012) recognized nine species including A. anceps(Lam.) Decne. Harvey et al. (2013) considered theirwork provided “a basis for future molecular-systemat-ics studies,” also stating “The correct application ofspecies names requires detailed analyses and compar-isons of diagnostic characters in type and modernmaterial. The types of many species of Amphiroa, how-ever, have not been examined in a modern contextand, as a result, the application of many names to spe-cies has been based largely on tradition and thus haslacked the nomenclatural foundation essential forstability.” The position of the recently described Litho-phyllum riosmenae relative to the geniculate Amphiroaspp. strongly suggests that this species belongs to agenus other than Lithophyllum.Recognition of rank within the coralline algae: criteria

for the separation of families and orders. In our analy-ses the families Rhodogorgonaceae and Corallina-ceae each receive 100% support under all methodsof analysis (Fig. 1). The position of the Rhodogor-gonaceae within the wider Corallinophycidae is notclearly resolved. The Sporolithaceae received mod-erate support (71/1) and the Hapalidiaceae wassupported as monophyletic (92/1).Le Gall et al. (2010), in analyses that resulted in

the establishment of the Sporolithales, includedthree species from the Corallinales - two from theCorallinaceae and a single species from the familyHapalidiaceae. Their analyses resolved the Sporo-lithaceae as being monophyletic, and they con-cluded that “in order to retain a system ofclassification within the Corallinophycidae based onnatural relationships options include transfer of theSporolithaceae from the Corallinales to the Rhodo-gorgonales, subsume the Rhodogorgonales withinan expanded Corallinales, or transfer the Sporolith-aceae to a new order.” They concluded that theRhodogorgonales and Corallinales were sufficientlydistinct in terms of genetic divergence and “mor-pho-anatomical differences” to warrant recognitionof distinct orders.The families of the Corallinophycidae all possess

pit plugs with two cap layers and with the outer capan enlarged dome-like layer. The families Rhodo-gorgonaceae, Sporolithaceae, Hapalidiaceae, andCorallinaceae are each characterized by particularreproductive and anatomical features, particularlythe arrangement and division of tetrasporangia, andthe presence or absence of apical plugs in concepta-cles.The data from our study presents us with a

dilemma – what weight should be placed on repro-ductive development in relation to rank? Do weapply the criteria used by Le Gall et al. (2010) inthe recognition of orders within the Corallinophyci-

10 WENDY A. NELSON ET AL.

dae? If the Sporolithaceae and Rhodogorgonaceaeare each to be recognized as orders on the basis ofgenetic differences and anatomical diagnostic char-acters, one argument is that the family Hapalidia-ceae also warrants recognition at the ordinal level.The Hapalidiaceae, resurrected by Harvey et al.(2003a) for taxa possessing zonately arranged tetra/bisporangia, with apical plugs, born in multiporatetetrasporangial conceptacles, is strongly supportedas being monophyletic with a sister-relationship withthe Corallinaceae, a result also found by Bittneret al. (2011). Under this scenario, it is highly likelythat Corallinapetra would be in a separate orderbased on both reproductive anatomy and geneticdifferences, as the anatomical and morphologicalcharacters of Corallinapetra do not align with currentfamily/order boundaries. The alternative option isto subsume the families Rhodogorgonaceae, Sporo-lithaceae, Hapalidiaceae and Corallinaceae into asingle order within the sub-class Corallinophycidae.The weight placed on reproductive development isthe critical issue. Does the recognition of at leastfour orders in the Corallinophycidae reflect thedivergence of clades from evolutionarily distinct lin-eages, and the ancient origins of these red algae, oris it an unnecessary proliferation of higher ranks?

In our view the erection of the order Hapalidialesfor this distinctive lineage of calcified algae is war-ranted in light of our current understanding of thephylogenetic relationships within the Coral-linophycidae and the possession of distinctive tet-rasporangial conceptacles.

Hapalidiales W. A. Nelson, J. E. Sutherl. and T. J.Farr et H. S. Yoon ord. nov.

Diagnosis: with the characteristics of the Coral-linophycidae (Le Gall and Saunders 2007); differsfrom other orders (Corallinales, Rhodogorgonales,Sporolithales) in producing zonately divided tetrasp-orangia, with tetra/bisporangia borne in concepta-cles, with apical plugs and developing beneathmultiporate plates, without genicula, and lackingsecondary pit-connections.

Type family: Hapalidiaceae Gray 1864: 22, emen-davit Harvey et al. 2003a: 995.The Hapalidiaceae currently includes three sub-

families: Choreonematoideae, Austrolithoideae, andMelobesioideae. The Hapalidiaceae was originallyestablished by Gray (1864) for a single monotypicgenus, Hapalidium roseolum K€utzing (1843: 385).Chamberlain (1983: 300) determined that Hapalidi-um roseolum was a heterotypic synonym of Melobesiamembranacea (Esper) J. V. Lamouroux, the type spe-cies of Melobesia. Although the family was not recog-nized by subsequent authors, Woelkerling (1988:86) noted that the family name Hapalidiaceae waslegitimate and thus available for a family thatincluded the genus Melobesia. Harvey et al. (2003a)determined that the family name Hapalidiaceae wasthe oldest available for this group when they resur-rected the family and emended its description. Dow-eld (2012) has proposed the conservation of thelater name Melobesiaceae against the two earliernames Hapalidiaceae, and Lithothamniaceae,arguing the earliest name, Hapalidiaceae, was “prac-tically forgotten” prior to its revised circumscriptionby Harvey et al. (2003a). Table 3 summarizes thecharacters of the four orders of the Corallinophyci-dae. In the light of the erection of the Hapalidiales,the original concept of the Corallinales requiresemending:Corallinales P.C. Silva et H.W. Johansen (1986, p.

250) emendavit W.A. Nelson, J.E. Sutherl., T.J. Farret H.S. YoonAs emended here, the Corallinales includes those

taxa posessing uniporate tetrasporangial concepta-cles, zonately divided tetrasporangia, lacking apicalplugs, with cell fusions or secondary pit connec-tions, and with pit plugs with 2 cap layers with theouter cap an enlarged dome-like layer.Future directions. As seen in this study, there are

important issues facing coralline taxonomy, withprogress seriously constrained by inadequate under-standing of generic and species concepts. Systematicresearch on coralline algae is benefiting greatly

TABLE 3. Characters of the four orders of Corallinophycidae.

Rhodogorgonales Sporolithales Hapalidiales Corallinales

Position oftetrasporangia

NA Calcifiedcompartments

Multiporateconceptacles

Uniporate conceptacles

Tetrasporangiadivision

None recorded:possiblecarpotetrasporangiain Renouxia

Cruciate Zonate Zonate

Apical plugs(at conceptaclepore)

NA Present Present Absent

Cell fusions andpit connections

No secondary pitconnections

Cell fusions andsecondary pitconnections

Cell fusions (not inChoreonematoideae)

Cell fusions or secondarypit connections

Pit plugs 2 cap layers with theouter cap an enlargeddome-like layer

2 cap layers withthe outer cap anenlarged dome-likelayer

2 cap layers withthe outercap an enlargeddome-like layer

2 cap layers with the outercap an enlarged dome-likelayer

MULTI-GENE ANALYSIS OF NZ CORALLINE ALGAE 11

from the application of molecular sequencing toolsand the phylogenetic analyses that these dataenable. Although accompanied by many technicalchallenges, the use of ancient DNA approaches andnext generation sequencing methods to obtain datafrom type material of red algae is yielding importantnew information, enabling for example, the exami-nation of fossil corallines (Hughey et al. 2008), aswell as the clarification of type concepts (summa-rized in Hughey and Gabrielson 2012, Hind et al.2014, Hughey et al. 2014). However, there areimpediments to progress: obtaining sequence datafrom type material requires not only access to her-barium material (with many herbaria reluctant toallow destructive sampling of type material), butalso access to facilities where appropriate ancientDNA protocols can be followed (e.g., Cooper andPoinar 2000, Hughey and Gabrielson 2012).Although Hughey and Gabrielson (2012) argue thatthe designation of epitypes with contemporary mate-rial has to be seen as a “last resort, not an alterna-tive to sequencing type material” and at the veryleast based on topotype material, it may be that theselection of epitypes accompanied by sequence datawill help to deal with the current bottlenecks thatare constraining progress in coralline taxonomicresearch.

The research reported here has revealed signifi-cantly greater diversity of coralline algae from theNew Zealand region than previously recognized,and joins other recent studies where intensive sam-pling of regional floras has been undertaken withsimilar results. It is clear to us that further research,involving targeted collection programs, multigenephylogenetic analyses and morpho-anatomical char-acterization, is needed before relationships anddiversity of the Corallinophycidae will be fullyunderstood.

The research has been supported by funding from the NewZealand Ministry for Primary Industries within the Biodiver-sity Research Programme (ZBD2001-05, ZBD2004-07,ZBD2009-03), as well as from NIWA within the National Cen-tre for Coasts & Oceans (Research Programme 2). This workwas also supported by grants from the Korean Rural Develop-ment Administration Next-generation BioGreen21(PJ009525) and National Science Foundation RedToL Project(DEB 1317114) to HSY. We also acknowledge the contribu-tion of the NeSI high-performance computing facilities at theUniversity of Auckland and the staff at NeSI and Centre foreResearch. New Zealand’s national facilities are provided bythe New Zealand eScience Infrastructure (NeSI) and fundedjointly by NeSI’s collaborator institutions and through theMinistry of Business, Innovation and Employment’s Infra-structure programme. URL: http://www.nesi.org.nz. Wewould like to thank the many collectors and field assistantswho contributed to these programs, and colleagues, particu-larly Paul Gabrielson, for valuable discussions.

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Supporting Information

Additional Supporting Information may befound in the online version of this article at thepublisher’s web site:

Table S1. Collection information for New Zea-land specimens used in this study.

Table S2. GenBank accession numbers for taxain the phylogenetic analyses.

MULTI-GENE ANALYSIS OF NZ CORALLINE ALGAE 15