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Systematics and Phytogeography |
2The Lewis B. and Dorothy Cullman Program for Molecular Systematics Studies, The New York Botanical Garden, Bronx, New York 10458 USA; 3Department of Botany, University of Queensland, Brisbane, Queensland 4072 Australia
Received for publication February 26, 2002. Accepted for publication May 2, 2002.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: Aldrovanda • carnivorous plants • Dionaea • DNA • Droseraceae • molecular systematics • phylogeny
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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called Dionaea muscipula Ellis (Venus' flytrap) "one of the most wonderful plants in the world" because of its rapidly closing, snap-trap leaves, which are used to capture and digest small animals for the plants' nutritional benefit. Its trapping mechanism is one of the fastest movements known among plants (Iijima and Sibaoka, 1985
; Juniper, Robins, and Joel, 1989
), and the monotypic genus is well known to both schoolchildren and scientists. Few people, however, are as familiar with the aquatic waterwheel plant (Aldrovanda vesiculosa L.), which employs a similar snap-trap mechanism for trapping prey underwater (Fig. 1).
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). The genus was named in honor of Ulisse Aldrovandi, a prominent Italian naturalist, and the specific epithet used by Linnaeus in 1753 refers to the fact that the leaves were originally thought to trap air for buoyancy (de Lassus, 1861
; Lloyd, 1976
). Darwin (1875)
carefully studied living specimens and suspected the plant, instead, to use its leaves for small animal capture, as was proven later to be the case. Aldrovanda is a rootless, submerged, aquatic herb that produces whorls of 7–8 leaves per node. It grows in dystrophic lakes and ponds throughout the Old World. In its habit and distribution, therefore, it is similar to many aquatic species of the carnivorous bladderworts (Utricularia spp.). Rather than producing underwater bladders for catching prey, however, the leaves of Aldrovanda consist of a pair of oval or round leaf lobes, 5–10 mm long, that resemble an open bivalve shell. On the inner surface of each lobe are approximately 20 hairs that serve as triggers for the snap-trap. This foliar morphology is so strongly reminiscent of the leaves of Venus' flytrap that Darwin (1875)
described Aldrovanda as "a miniature, aquatic Dionaea."
In its reproductive morphology, however, Aldrovanda shares more characters with the carnivorous sundews (Drosera spp.) than it does with Dionaea. A comparison of these taxa is presented in Table 1. Both Aldrovanda and Drosera have flowers with five stamens and parietal placentation of their ovules, whereas the flowers of Dionaea have 15 stamens and basal placentation. The pistil of Aldrovanda has separate, undivided styles like some species of Drosera, whereas the styles of Dionaea are completely united. Both Dionaea and Drosera display multiple operculate pores around the connection area of their pollen tetrads (Takahashi and Sohma, 1982
). Aldrovanda also sheds its pollen as tetrads, but its pollen is triporate and differs from both Dionaea and Drosera. Consequently, Nakai (1949)
proposed a monotypic family, Aldrovandaceae, to accommodate this enigmatic taxon rather than to classify it in Droseraceae.
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). Both Aldrovanda and Drosera have mostly small chromosomes with no primary constriction and 2–4 CMA+ DAPI– sites. In contrast, the chromosomes of Dionaea are all medium in size with localized centromeres and show no CMA+ DAPI– sites.
The only cladistic study of phylogenetic relationships within Droseraceae that sampled Aldrovanda included 14 morphological characters for this taxon (Williams, Albert, and Chase, 1994
). The results showed a sister relationship between Aldrovanda and Drosera, with Dionaea sister to the pair, implying that plants with adhesive, glandular flypaper-traps may have evolved from plants with snap-traps. This scenario seems unlikely because there is evidence (Williams, 1976
; Juniper, Robins, and Joel, 1989
) that the trigger hairs of Dionaea are derived from glandular trichomes, not vice versa. On the other hand, a number of molecular phylogenetic studies have demonstrated that carnivory evolved independently among several different lineages of flowering plants (Albert, Williams, and Chase, 1992
; Meimberg et al., 2000
). Furthermore, convergent evolution has selected for similar trapping mechanisms among these unrelated lineages. For example, leaves used as pitfall-traps (i.e., "pitcher plants") are known to have evolved within lineages of the orders Caryophyllales, Oxalidales, Ericales, and Poales (sensu APG, 1998
). Likewise, flypaper-traps have evolved in at least three or four different clades.
The exact relationship between Aldrovanda, Dionaea, and Drosera has not been addressed adequately, and molecular data clearly have a role to play in sorting out this problem (Givnish, 1989
). With full knowledge that similar trapping mechanisms have evolved independently among different lineages of carnivorous plants, we set out to determine the phylogenetic position of Aldrovanda using DNA sequence data from both nuclear and plastid genes. The placement of this taxon among angiosperms has implications not only for taxonomy, but also for determining whether snap-traps evolved more than once among angiosperms and whether they were derived from flypaper-traps or vice versa.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) by analyzing its rbcL, atpB, and 18S sequences within a large, published matrix of angiosperm sequences that included representatives of most eudicot orders (modified from Soltis et al., 2000)
. Based on this result and on previously published, broadscale analyses of Caryophyllales (Lledó et al., 1998
; Meimberg et al., 2000
; Soltis et al., 2000
; Cuénoud et al., 2002
), we sampled 15 species from 13 genera and nine families (Ancistrocladaceae, Dioncophyllaceae, Droseraceae, Frankeniaceae, Nepenthaceae, Plumbaginaceae, Polygonaceae, Simmondsiaceae, and Tamaricaceae) to represent the ingroup and appropriate outgroup taxa. The entire aligned matrix is available from the first author or online from The New York Botanical Garden (http://www.nybg.org).
Total DNA was extracted using the FastPrep (Qbiogene, Carlsbad, California, USA) and glassmilk method from approximately 0.5 cm2 of dried tissue as described by Struwe et al. (1998)
. Target loci were amplified in 50-µL volumes using standard polymerase chain reaction (PCR) protocols that included the addition of bovine serum albumin (BSA) and betaine. Amplification and sequencing primers were the same as used by Soltis et al. (2000)
except for matK, which we amplified and sequenced using primers 390F and 1326R as cited in Cuénoud et al. (2002)
. This resulted in 
900 base pairs (bp) of sequence for that gene. In all cases, resulting PCR products were purified using QIAquickTM spin columns (Qiagen, Valencia, California, USA) according to the manufacturer's protocols. Cycle sequencing reactions were completed using a combination of purified PCR template, primer, and dRhodamine Ready Reaction mix (Applied Biosystems, Foster City, California, USA) for 20 cycles. These reactions resulted in nearly complete, overlapping, forward and reverse strands of the target loci. Centri-Sep sephadex columns (Princeton Separations, Adelphia, New Jersey, USA) were used according to the manufacturer's instructions to remove excess dye terminators and primer from the cycle sequencing products. These were subsequently dehydrated, resuspended in a mixture of formamide and loading dye, and pipetted onto a 5% denaturing polyacrylamide gel. Samples were run on an Applied Biosystems ABI 377XL automated DNA sequencer, and resulting electropherograms were edited using Sequencher 3.0 (GeneCode, Ann Arbor, Michigan, USA).
Phylogenetic analyses
The individual and combined data matrices were analyzed using the parsimony criterion in PAUP* version 4.0b8 (Swofford, 2001
). Simmondsia was specified as the single outgroup taxon based on the topologies produced in broader phylogenetic studies (e.g., Soltis et al., 2000
; Cuénoud et al., 2002
). The 5' and 3' ends of each gene sequence (typically 30–50 bp) were excluded to minimize missing data. In addition, a hypervariable 32-bp region of matK located at 190 bp from the 5' end was excluded because of ambiguous alignment. All equally parsimonious trees were found by executing a branch and bound search with gaps treated as missing data and characters unordered and weighted equally. Internal support was determined by performing parsimony bootstrap analyses (1000 replicates, simple addition, and tree bisection-reconnection [TBR] branch swapping). Although the phylogenetic position of Drosera regia Stephens was found to be inconsistent among the individual gene analyses (i.e., either sister to the other species of Drosera or sister to the Aldrovanda-Dionaea clade), no bootstrap support >50% (i.e., "hard" incongruence sensu Weins, 1998
) was detected among the individual trees, so combination of the four data sets proceeded. To address the evolution of trapping mechanisms, we used both ACCTRAN and DELTRAN optimizations in MacClade version 3.04 (Maddison and Maddison, 1992
) to examine the distribution of the following character states: not carnivorous, pitfall-trap, flypaper-trap, and snap-trap.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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), even when Dionaea is elevated to familial status as Dionaeaceae (Small, 1933
). In fact, the illegitimate combination Drosera aldrovanda F. Muell. has even been made (von Mueller, 1876
) to accommodate Aldrovanda within Drosera rather than to treat it as a distinct genus. In contrast, Dionaea has never been classified under any other generic name.
The common origin and evolution of snap-traps
Until now, no molecular data have been available to help settle the ongoing debate regarding the exact phylogenetic position and classification of Aldrovanda vesiculosa. However, our results show that Aldrovanda is sister to Dionaea, not to Drosera, and that snap-traps evolved only once among carnivorous plants (moving Aldrovanda away from Dionaea to a position sister to Drosera requires an additional ten steps). We have pointed out the numerous morphological differences between Aldrovanda and Dionaea, but there are other likely synapomorphies in addition to the common trapping mechanism that unite them. Boesewinkel (1989)
considered the ovules and seeds of Aldrovanda to be more similar to those of Dionaea than to any other taxon in Droseraceae. The two genera also share a similar trichome/gland morphology, including sessile and stellate glands lacking vascular tissue and multicellular trigger hairs (Juniper, Robins, and Joel, 1989
). Moreover, the traps of both Aldrovanda and Dionaea function in a similar manner by rapid transmission (6–17 cm/s) of action potentials between excitable cells (Iijima and Sibaoka, 1985
). Some species of Drosera also generate action potentials, but their trasmission velocity is 10% slower. Finally, both Aldrovanda and Dionaea lack 7-methyljuglone, but possess plumbagin in their tissues (Culham and Gornall, 1994
). However, there are several species of Drosera that have this same napthoquinone profile.
Based on a number of shared morphological characters and the structure of the molecular cladogram, therefore, we recommend the recognition of Aldrovanda, Dionaea, and Drosera, but not Drosophyllum, as members of Droseraceae s.s. Drosophyllum is a monotypic genus from Portugal. Although it traps insects with glandular trichomes, it differs from Drosera in a number of morphological features, and its disassociation from Droseraceae s.s. has been documented and discussed in detail by others (Williams, Albert, and Chase, 1994
; Meimberg et al., 2000
). It should be treated as the sole member of Drosophyllaceae.
Furthermore, based on this cladogram, it is most parsimonious to hypothesize that the snap-traps of Aldrovanda and Dionaea were derived from a common terrestrial ancestor that had flypaper-traps, not the other way around. Likewise, the pitfall-traps of the tropical pitcher plant genus Nepenthes also must have evolved from an ancestor with adhesive, flypaper traps. Its exact position among the genera of Caryophyllales has been ambiguous to date; Albert, Williams, and Chase, (1992)
and Fay et al. (1997)
found Nepenthes to be sister to a Plumbago/Rheum clade, Williams, Albert, and Chase (1994)
and Lledó et al. (1998)
found it sister to the entire carnivorous clade, Meimberg et al. (2000)
to the Drosophyllum/Ancistrocladus/Dioncophyllaceae clade, and Cuénoud et al. (2002)
to Droseraceae. Only one of these relationships—that of Meimberg et al. (2000)
, which is the same as found here—has been supported by the bootstrap.
Lledó et al. (1998)
and others have discussed the hypothesis that the common ancestor of this mostly insectivorous, caryophyllid clade was preadapted for a carnivorous lifestyle because many of the noncarnivorous, outgroup taxa (e.g., Plumbago, Tamarix, Frankenia) possess multicellular glands that secrete mucilage, salt, or other compounds. Modification of these glands for animal capture and/or digestion followed by their co-option as snap-trap trigger hairs in the ancestor of Dionaea and Aldrovanda seems plausible and has been suggested (Juniper, Robins, and Joel, 1989
; Williams, 1976
). The habitat of Dionaea muscipula frequently floods (Roberts and Oosting, 1958
). Yet even under these conditions, it has been reported that the flytraps continue to grow and capture prey underwater (Schnell, 1976
). Thus, it is not difficult to envision a Dionaea-like, terrestrial ancestor of Aldrovanda vesiculosa becoming adapted to a permanently aquatic lifestyle as suggested by Arber (1920)
.
Implications for biogeography and conservation
Aldrovanda has a fossil pollen and seed history dating back to the lower Tertiary (von Kircheimer, 1941
; Muller, 1981
; Yakubovskaya, 1991
), with at least 13 different names having been applied to these fossils (e.g., A. siberica V. Nikit., A. europaea Negru, and A. praevesiculosa Kirchh.). Dionaea also has a documented fossil history, with the discovery of pollen from the middle Miocene and Pliocene of central Europe that is comparable to modern Dionaea muscipula (Muller, 1981
). These microfossils indicate that both genera were once more common and widespread in distribution than they are today.
The ancient seeds of Aldrovanda also show enough morphological variation for Yakubovskaya (1991)
to have hypothesized an evolutionary continuum along at least two ancestral lines. One of these, originating from A. ovata M. Chandl., has become extinct; the other, from A. intermediata E. Reid and M. Chandl., ultimately led to the evolution of the modern species. Moreover, these fossils strongly support a date of origin for this entire, highly specialized, carnivorous lineage of at least 65 million years ago and probably much earlier. This age may assist in explaining the current transcontinental distributions and putative Gondwanan origins of Drosera (Meimberg et al., 2000
) and Nepenthes (Meimberg et al., 2001
).
Modern Aldrovanda vesiculosa is a relictual species historically distributed in parts of western Europe, central Africa, southern India, Japan, and Queensland, Australia. It is now presumed to be extinct in several countries (e.g., France, Italy, and India), and is shrinking drastically in the number and size of its extant populations (Adamec, 1995
; Kaminski, Adamec, and Breckpot, 1996
; Kundu, Basu, and Chakraverty, 1996
). For 200 yr, it had been recorded (Adamec and Lev, 1999
) from more than 150 sites within Europe. Today it is estimated to survive in fewer than 36 localities, most of which are in Poland, Ukraine, and Russia (Adamec, 1995
). For this reason, it is now considered one of the rarest aquatic plants in the Old World (Kaminski, Adamec, and Breckpot, 1996
). Likewise, Dionaea muscipula can be considered a relictual species with a narrow, endemic distribution of less than 300 km2 in the southeastern United States.
Both Aldrovanda and Dionaea are severely threatened by anthropogenic disturbance of their specialized habitats (Adamec, 1995
; Kaminski, Adamec, and Breckpot, 1996
; Kundu, Basu, and Chakraverty, 1996
), and Dionaea has been overcollected as a horticultural curiosity; it is currently listed on Appendix II of CITES. Recent reintroductions of Aldrovanda into the Czech Republic (Adamec and Lev, 1999
) appear to be successful and offer hope of conserving this remarkable species. Introductions of Dionaea have also taken place in Florida, New Jersey, and Deleware (USDA NRCS, 2001
), but the future survival of these two species in their native habitats is of serious concern to conservationists. It is our hope that the fundamental systematic data presented here have helped to clarify the classification, phylogenetic relationships, evolutionary origin, and ecological adaptation of what Arthur Dobbs (Dillwyn, 1843
), the former Governor of North Carolina and discoverer of Venus' flytrap, called "the great wonder of the vegetable kingdom."
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FOOTNOTES |
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4 Author for reprint requests (kcameron{at}nybg.org
)
5 Current address: Laboratory of Molecular Systematics, Museum Support Center A2000, Smithsonian Institution, Washington, D.C. 20560 USA
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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