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The phylogenetic position of subfamily Monotropoideae (Ericaceae) inferred from large ribosomal subunit (26S) rRNA gene DNA sequences.

INTRODUCTION

Obligate myco-heterotrophs are non-green plants that obtain fixed carbon from fungi (Bidartondo and Bruns, 2002). Such plants have arisen independently in many plant families including the Orchidaceae, Burmanniaceae, Corsiaceae, Scropulariaceae and Gentianaceae. Several closely related mycoheterotrophic species occur in subfamily Monotropoideae of family Ericaceae. According to Kron et al. (2002), the Monotropoideae consists of an autotrophic tribe, Pyroleae, and two mycoheterotrophic tribes, Pterosporeae and Monotropeae. However, there has been little consensus concerning the circumscription or phylogenetic position of this subfamily. For example, some authors placed the autotrophic members in family Pyrolaceae and the mycoheterotrophic members in family Monotropaceae (e.g. Rydberg, 1914; Small, 1914; Cronquist, 1981; Anderberg, 1992). Other authors combined the two groups in family Pyrolaceae (e.g. Copeland, 1939, Lawrence, 1965) and others have positioned both groups within Ericaceae (e.g. Copeland, 1941, 1947, Wood, 1961, Stevens, 1971, Wallace, 1974, Takhtajan, 1980, Judd and Kron, 1993).

Aphylogeny inferred from partial 28S rRNA gene sequences by Cullings (1994) indicated that the myco-heterotropic Monotropoideae are polyphyletic. However, due to possible misidentification of specimens used in that analysis, Cullings (2000) stated that no taxonomic conclusions regarding members of the Monotropoideae should be drawn from those data.

A phylogenetic analysis of rbcL sequences by Kron and Chase (1993) suggested that the Monotropoideae are positioned between the basally diverged Enkianthus and all remaining representatives of Ericaceae. A phylogenetic study using 18S rRNA sequences separately and combined with rbcL sequences by Kron (1996) recovered topologies that suggested various positions for the Monotropoideae with Ericaceae. Results from that study also suggested that the monotropoideae are monophyletic but that the myco-heterotrophic members may form a paraphyletic group. From their phylogenetic analysis of Ericaceae using both morphological data and matK, and rbcL and 18S molecular data, Kron et al. (2002) concluded that the subfamily monotropoideae is composed of the autotrophic tribe Pyroleae and two mycoheterotrophic tribes Monotropeae and Pterosporeae. In that study, the Enkianthoideae are positioned as the most basally diverged subfamily followed by the Monotropoideae which are sister to all remaining subfamilies in Ericaceae. However, the analysis by Kron et al. (2002) was unable to resolve the relationships among the tribes of the Monotropoideae.

The purpose of this study is to investigate the phylogenetic relationship of subfamily Monotropoideae with family Ericaceae. This Relationship is inferred from a maximum parsimony analysis using large ribosomal subunit (26S) rRNA gene DNA sequences.

MATERIAL AND METHODS

Scientific name, voucher information, and GenBank accession numbers for the taxa analyzed in this study are listed in Table 1. Based on the studies by Bremer et al. (2002) and Anderberg et al. (2002) representatives of Cyrillaceae are designated as outgroup (Table 1). An approximate 1 kb DNA segment of the 26S gene was sequenced for the taxa included in this analysis. Spanning base positions 4-969 in Oryza sativa (Sugiura et al., 1985), this segment is characterized by conserved segments and more variable expansion segments (Kuzoff et al., 1998).

Total DNA was extracted from tissue using the CTAB method of Doyle and Doyle (1987). DNA sequences were amplified via polymerase chain reaction (PCR) (Mullis and Faloona, 1987; Saiki et al. 1988) from total DNA extracted for the species listed in Table 1 with combinations of forward and reverse primers referenced in Neyland (2002). Amplification was achieved with Tfl enzyme (Epicentre Technolgies, Madison, WI), using the following thermocycling protocol: a hot start at 94[degrees]C for 1 minute, 55[degrees]C for 1 minute; 72[degrees]C for 3.5 minutes, a terminal extension phase at 72[degrees]C and an indefinite terminal hold at 4[degrees]C. The double stranded PCR product was purified with FQIAquick (Qiagen, Hilden, Germany) using the manufacturer's protocol. Two [micro]l of each sample were electrophoresed in a 1.0% agarose mini-gel for quantification against a known standard. Automated sequencing was conducted on an ABI Prism 377 Sequencer with XL Upgrade (hosed at Louisiana State University, Baton Rouge, LA, USA) using ABI Prism, Big Dye Terminator cycle sequencing protocol (P.E. Applied Biosystems, Foster City, CA, USA) Sequences have been deposited in the GenBank database (Table 1).

Phylogenetic analyses were performed using the heuristic search algorithm with Phylogenetic Analysis Using Parsimony (PAUP) version 4.0b10 software (Swofford, 2002). A parsimony search of 1000 random stepwise addition replications was performed.

Transition/transversion rates were calculated using MacClade software (Maddison and Maddison, 2003). All characters, including transversions and transitions, were weighted equally. Gaps were treated as missing data. As a measure of clade stability or roubustness, bootstrap support (Felsenstein, 1985; Sanderson, 1989) was calculated. Ten thousand full heuristic bootstrap replications were employed in this analysis.

RESULTS

Sequences were easily aligned by eye. Gaps were introduced to accommodate point insertions/deletions (INDELS) in the data set. INDELS could not be determined unequivocally to be homologous and, therefore, were not treated as characters. The heuristic search resulted in a single most parsimonious tree (Fig. 1) of 388 steps with a consistency index of 0.6624 and a retention index of 0.5691.

Absolute distances within the entire data set range from a minimum of 8 between Archeria and Epacris and a maximum of 100 between Monotropa hypopithys and Harrimanella. Transitions numbered 247 and transversions numbered 80. Therefore, the ration of transitions to transversions is about 3:1.

[FIGURE 1 OMITTED]

DISCUSSION

The findings of this study suggest that the Monotropoideae are derived within Ericaceae. The relationships among the Monotropoideae tribes received either moderate or strong bootstrap support as indicated by the phylogram (Fig. 1). This recovered topology supports Kron et al's (2002) taxonomy in which tribes Pyroleae, Pterosporeae and Monotropeae comprise a monophyletic group.

The derived position of the Monotropoideae in the phylogram (Fig. 1) supports the hypothesis of Henderson (1920) who suggested that these plants represent the end products of autotrophic members of Ericaceae and that of Furman and Trappe (1971) who considered them occupants of an advanced stage of evolution. However, support for the position of Monotropoideae has little bootstrap support (Fig.1). If all nodes that received less than 50% bootstrap support were collapsed, the Enkianthoideae would be the most basally diverged subfamily in Ericaceae followed by the Arbutoideae (Fig. 1). The basal position of the Enkianthoideae in this study agrees with that of Kron et al. (2002). However, the position of the Arbutoideae as sister to remaining subfamilies of Ericaceae in this study contrasts with the phylogeny recovered in the study by Kron et al.(2002) that suggested the Monotropoideae occupy this position. Perhaps the difference between the two studies is due primarily to the type of sequence data used. Kron et al. (2002) used chloroplast matK, and rbcL sequences and nuclear-encoded ribosomal 18S sequences. Because the chloroplast genomes of plants that no longer carry on photosynthesis are subject to reduced selection pressures, their mutation rates are often elevated which can lead to homoplasy and misleading phylogenies (Felsenstein, 1978; Nickrent and Star, 1994; dePamphilis, 1995; Nickrent and Duff, 1996, 1996; Nickrent et al. 1998; Chase et al., 2000; Neyland, 2001, 2002; Neyland and Hennigan, 2003). Branches with excess substitutions will tend to share spurious synapomorphies with other long branches and, therefore, group with them, even if they are not closely related (dePamphilis, 1995). Therefore, the use of chloroplast sequences in constructing phylogenies that include obligate mycoheterotrophic plants is problematic. Kron et al. (2002) did use some nuclear-encoded 18S sequences in their analysis. However, the sole representative of the Monotropoid included in their 18S data was that of the autotrophic Pyrola rotundifolia of the tribe Pyroleae. Therefore, Kron et al.'s (2002) study included no nuclear-encoded sequences from the myco-heterotrophic members of the Monotropoideae. It is not surprising that phylogenies constructed from different genomes would produce different results, especially when obligate myco-heterotrophic plants are included.

The relationship of the tribes within the Monotropoideae remains equivocal. Although a large amount of data has been acquired and analyzed in this and other studies, there remains no robust support for any particular hypothesis. Perhaps the major problem with recovering a well-supported phylogenetic hypothesis of this group is a result of the high mutation rates associated with mycoheterotrophy. Although the case was made previously that reduced selection pressures on chloroplast genomes often leads to higher mutation rates and misleading phylogenies, results from this study also suggest that higher mutation rates may be associated with nuclear-encoded genomes as well. Specifically, the branch lengths, which are direct reflection of high mutation rates, for both tribes Monotropeae and Pterosporeae are comparatively long (Fig. 1). Therefore, the effects of long-branch attraction also may play a role in the recovered phylogeny of the present study. Thus, the systematic of the Monotropoideae remains a perplexing problem and is worthy of further research.

ACKNOWLEDGMENTS

We thank Jack Alexander and Tom Ward of the Arnold Arboretum, Harvard University, Cambridge, MA, USA; Christopher Quinn, University of New South Wales, Sydney Australia; Richard and Jessie Johnson, Briarwood Estate, LA, USA; Martin Bidartondo, University of California, Berkeley, CA, USA; Darren Crayn, Royal Botanic Garden, Sydney, Australia and Kristin Wilson, McNeese State University, Lake Charles, LA, USA for their assistance. Funding was provided by the senior author by the College of Science, McNeese State University.

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Ray Neyland (1) * and Mark Merchant (2)

(1) Department of Biology and Allied Health, McNeese State University Lake Charles, LA 70609.

(2) Department of Chemistry, McNeese State University, Lake Charles, LA 70609.

* Corresponding Author: Ray Neyland: rneyland@mcneese.edu
Table 1. Taxa used in this study. All in group members are from
Ericaceae (sensu Kron et al.2002). Outgroup members are from
Cyrillaceae. Voucher/accession data re give. Those taxa collected by
Neyland are housed at the McNeese State University herbarium (MCN) that
by N. G. Miller is housed by at New York State Museum (NYS). The
voucher supplied by the Arnold Arboretum, Harvard is designated by the
prefix AAH. Vouchers for DNA extracts supplied by The Royal Botanic
Gardens, Sydney and The Royal Botanic Gardens, Edinburgh are housed at
the University of New South Wales (UNSW) and The Royal Botanic Garden
Edinburgh (E) respectively. Frozen Tissue from which the DNA extract of
Sarcodes sanguinea was derived is maintained at the Department of
E.S.P.M., University of California, Berkley.

Taxon                                   Voucher/Accession

Ingroup

Subfamily Monotropoideae
Tribe Monotropeae
Monotropa hypopihtys L.                 Neyland 2037
Monotropa uniflora L.                   Neyland & Hennigan 1954
Tribe Pyroleae
Chimaphila maculate (L.) Porsh          Neyland 2049
Moneses uniflora (L.) Gray              Neyland 2079
Tribe Pterosporeae
Pterospora andromedea Nutt.             Neyland 2078
Sarcodes sanguinea Torr.                --

Subfamily Enkianthoideae

Enkianthus campanulatus (Miq.)          Neyland 2125
Nicholson

Subfamily Arbutoideae
Arbutus unedo L.                        E19810674
Arctostaphylos uva-ursi (L.) Spreng     Neyland 2094
Subfamily Ericoideae
Tribe Ericaea
Erica carnea L.                         Neyland 2092
Tribe Phylloidoceae
Kalmia latifolia L.                     Neyland 1905
Tribe Empetreae
Corema conradii (Torr.) Loudon          AAH 795-90-B
Tribe Rhodoreae
Rhododendron canescens (Michx.)         Neyland 659
Sweet
Subfamily Cassiopoideae
Cassiope fastigiata (Wall.) D.Don       E19842198
Subfamily Harrimanelloideae
Harrimanella hypnoides (L.) Coville     N.G. Miller 10974
Subfamily Styphelloideae
Tribe Archarieae
Archeria racemosa Hook. F.              UNSW23608
Tribe Epacrideae
Epacris lanuginose Labill.              UNSW 22531
Subfamily Vaccinoideae
Tribe Andromedeae
Andromeda polifolia L.                  E19772596
Tribe Vaccinieae
Vaccinium elliottii Chapm.              Neyland 1189
Outgroup
Cliftonia monophylla (Lam.)Britt. Ex    Neyland 2093
Sarg.
Cyrilla racemiflora L.                  Neyland 856

Taxon                                   GenBank
                                        Accession

Ingroup

Subfamily Monotropoideae
Tribe Monotropeae
Monotropa hypopihtys L.                 AF543835
Monotropa uniflora L.                   AF540062
Tribe Pyroleae
Chimaphila maculate (L.) Porsh          AY294625
Moneses uniflora (L.) Gray              AY566296
Tribe Pterosporeae
Pterospora andromedea Nutt.             AY368156
Sarcodes sanguinea Torr.                AY737249

Subfamily Enkianthoideae

Enkianthus campanulatus (Miq.)          AY804243
Nicholson

Subfamily Arbutoideae
Arbutus unedo L.                        DQ067894
Arctostaphylos uva-ursi (L.) Spreng     AY596455
Subfamily Ericoideae
Tribe Ericaea
Erica carnea L.                         DQ065768
Tribe Phylloidoceae
Kalmia latifolia L.                     AY856380
Tribe Empetreae
Corema conradii (Torr.) Loudon          AY942693
Tribe Rhodoreae
Rhododendron canescens (Michx.)         AY561837
Sweet
Subfamily Cassiopoideae
Cassiope fastigiata (Wall.) D.Don       AY942692
Subfamily Harrimanelloideae
Harrimanella hypnoides (L.) Coville     DQ065769
Subfamily Styphelloideae
Tribe Archarieae
Archeria racemosa Hook. F.              AY870406
Tribe Epacrideae
Epacris lanuginose Labill.              DQ065767
Subfamily Vaccinoideae
Tribe Andromedeae
Andromeda polifolia L.                  DQ065770
Tribe Vaccinieae
Vaccinium elliottii Chapm.              AY561835
Outgroup
Cliftonia monophylla (Lam.)Britt. Ex    AY561839
Sarg.
Cyrilla racemiflora L.                  AY561838
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Author:Neyland, Ray; Merchant, Mark
Publication:Journal of the Mississippi Academy of Sciences
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Geographic Code:1USA
Date:Apr 1, 2011
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