The phylogenetic position of subfamily Monotropoideae (Ericaceae) inferred from large ribosomal subunit (26S) rRNA gene DNA sequences.
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.
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]
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.
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: firstname.lastname@example.org
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|
|Date:||Apr 1, 2011|
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