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OPEN ACCESS JCTS Article SERIES B

Creation and Carnivory in the Pitcher of Nepenthaceae and

R.W. Sanders and T.C. Wood

Core Academy of Science, Dayton, TN

Abstract The morphological adaptations of carnivorous plants and taxonomic distributions of those adaptations are reviewed, as are the conflicting classifications of the plants based on the adaptations, reproductive morphology, and DNA sequences. To begin developing a creationist understanding of the origin of carnivory, we here focus specifically on pitcher plants of Nepenthaceae and Sarraceniaceae because their popularity as cultivated curiosities has generated a literature resource amenable to baraminological analysis. Hybridization records were augmented by total nucleotide differences to assess similarities. Nonhybridizing species falling within the molecular range of hybridizing species were included in the monobaramin of the hybridizing species. The combined data support each of the three genera of the Sarraceniaceae as a monobaramin, but the three could not be combined into a larger monobaramin. With the Nepenthaceae, the data unequivocally place 73% of the species in a single monobaramin, strongly suggesting the whole (and, thus, family) is a monobaramin. The lack of variation in the carnivorous habit provides no evidence for the intrabaraminic origin of carnivory from non-carnivorous plants. An array of fascinating symbiotic relationships of pitchers in some species with unusual bacteria, insects, and vertebrates are known and suggest the origin of carnivory from benign functions of the adaptive structures. However, these symbioses still do not account for the apparent complex design for carnivory characteristic of all species in the two families.

Editor: J.W. Francis Received January 24, 2012; Accepted September 10, 2016; Published September 26, 2016

Introduction must be invoked by conventional biology to explain either the highly similar carnivorous adaptations, the flowers and , or Genesis 1:29-30 indicates that God gave plants to animals and the known nucleotide sequences among disparate carnivorous people for food, but today there are over 600 species of carnivorous groups (Soltis et al. 2005, p. 256 ff.). For example, Ellison plants that “eat” animals for food. All species produce modified and Gotelli (2009) claimed that plant carnivory has evolved or stems (“traps”) that capture and digest small animals independently six different times. (mostly arthropods) as a supplementary source of nitrogen, the The challenge for creation biology is to determine the benefits of which vary according to species (Ellison 2006). The created kinds, i.e., baramins, to which these plants belong and origin of these structural and chemical specializations challenges how such plants came to contravene God’s apparent pre-Fall both conventional and creationist science. Phylogenetic design. Specifically, baraminological analysis can assist us in relationships as determined by DNA sequence analysis do not understanding the origins of plant carnivory, either as the original support a single monophyletic origin of plant carnivory, despite design (or modification of that design) encompassing entire the sometimes startlingly similar morphologies and biochemistry baramins or as post-Fall specializations arising in limited species of the insect trapping and digesting structures. Thus, homoplasy or lineages within baramins.

©2016 The authors. This article is open access and distributed under a Creative Commons Attribution License, which allows unrestricted use, distribution, and repro- duction in any medium as long as the original author and medium are credited. Citation: Sanders and Wood. 2016. Creation and Carnivory in the Pitcher Plants of Nepenthaceae and Sarraceniaceae. Journal of Creation Theology and Science Series B: Life Sciences 6:70-80. The structurally and physiologically diverse traps can be Cunoniaceae, and the Byblidaceae are transported to the Lamiales categorized as pitchers, flypaper, snap traps, bladder traps, (Asteridae) between the Lentibulariaceae and Martyniaceae. As or corkscrew (also called lobster-pot) traps (Pietropaolo a result of these realignments, conventional biology proposes six and Pietropaolo 1986). The systematic occurrence of these origins of carnivory (Ellison and Gotelli 2009). In the case of the trap types among the eleven known families , flypaper traps are supposed to have given rise to with carnivorous species is of great interest both in terms of pitchers in the Nepenthaceae, snap traps in the , and occurrence within families and taxonomic placement of the a reversal to noncarnivory (two genera of the families. Several of the families are monogeneric with one or and Ancistrocladaceae sister to it). In the , pitchers few species; the Nepenthaceae are monogeneric with numerous and flypaper traps evolved independently in the Sarraceniaceae species; the Martyniaceae, Dioncophyllaceae, and Bromeliaceae and Roridulaceae, respectively. In the Lamiales, flypaper traps have only a few carnivorous species; and the Sarraceniaceae are (Martyniaceae, Byblidaceae, ) gave rise to bladders composed of three carnivorous genera. In all these cases, only and corkscrews either independently or in sequence in one trap type is found per family. However, in the Droseraceae and . Thus, evolutionary analyses posit three origins of and Lentibulariaceae, each composed of three genera, two and flypapers, which give rise to four other types, and four origins of three trap types occur, respectively, and each genus has only one pitchers. trap type. Flypaper traps are found in the Byblidaceae (1 sp.), To begin developing a creationist understanding of the origin Dioncophyllaceae (1 of 3 spp.), Droseraceae (, 110 spp.), of plant carnivory, we here focus specifically on pitcher plants. Drosophyllaceae (1 sp.), Lentibulariaceae (Pinguicula, 50 spp.), In the Nepenthaceae, Sarraceniaceae, and Cephalotaceae, pitcher Martyniaceae (9 of 13 spp.), and Roridulaceae (1 sp.). Snap traps traps usually form from the basal or apical portion of a that are limited to and Dionaea in the Droseraceae (2 spp.), develops as a tube rather than a flattened structure. However, in while bladders and corkscrews are unique to Utricularia (200 spp.) the Bromeliaceae an upright whorl of overlapping leaves can also and Genlisea (15 spp.), respectively, both in the Lentibulariaceae. form a tube, which is clearly non-homologous with those in the Pitchers occur in the Nepenthaceae (>130 spp.), Sarraceniaceae other families. The base of the tube fills with water that contains (25 spp.), Cephalotaceae (1 sp.), and Bromeliaceae (only 3 of either digestive enzymes produced by glands on the basal internal the 2110 spp.). The three species in the Bromeliaceae are the only of the pitcher or digestive bacteria unaided by plant- that are carnivorous; all other carnivorous plants produced enzymes. Above the water, the internal walls are waxy are herbaceous dicotyledons. and/or covered with downward-pointing hairs. Above that the Traditional classifications of these families, based on their pitcher may have light colored spots (windows), nectaries, or atypical flowers and vegetative bodies, are contradictory but have perfume glands to attract insects. The pitcher often has a flattened been eclipsed recently by molecular phylogenies (Soltis et al. 2005, portion of leaf distal to and overhanging the tube to prevent rain pp. 256ff). On the basis of presumed homology of diverse trap forms from diluting the water in the reservoir. The insects enter the top and similarity, Cronquist (1981, pp. 367, 368) united the of the trap to feed or look for food, find it hard to fly or crawl back Nepenthaceae, Sarraceniaceae, Droseraceae and Drosophyllaceae out, and slide down the tube into the digestive reservoir where as an order in his subclass Dilleniidae near the Theales. Takhtajan they drown. (1980, pp. 278-283) also considered the four to be close along These families occupy distinct, mostly separate, non- with the Cephalotaceae, Byblidaceae, and Roridulaceae near overlapping geographic areas. follicularis, the sole the Saxifragales in his subclass Rosidae. Similarly, Cronquist species of the Cephalotaceae, is found in southwestern Australia (1981, pp. 553-555, 569, 570) treated the latter three near the (Cronquist, 1981, p. 570). Nepenthaceae consist of the single genus Saxifragaceae and Pittosporaceae in his Rosales. Both placed the with >130 species native to the Old World tropics, from Dioncophyllaceae near the Theales and Violales in the Dilleniidae Madagascar to northeastern Australia (Cronquist 1981, p. 374) . (Takhtajan 1980, p. 271; Cronquist 1981, p. 407). Thorne (1976) Sarraceniaceae consist of three genera: (8 species), placed the Sarraceniaceae, Dioncophyllaceae, and Nepenthaceae (24 species), and the monospecific Darlingtonia. in separate suborders of his Theales in a superorder more or less Sarracenia species are native to of North America, and equivalent to the Dilleniidae, but separated the Droseraceae and is found only in northern , Drosophyllaceae to near the Saxifragaceae in his Rosales not far Oregon, and British Columbia. The species of Heliamphora grow from Byblidaceae and Roridulaceae in the Pittosporales. There on and around the tepuis of the Guiana Highlands of northern has been little disagreement about placing the Martyniaceae and (Cronquist 1981, p. 372; McPherson 2007, pp. 4, Lentibulariaceae in or near the Scrophulariales/Lamiales of the 22, 71, 77, 97, 102, 190, 191). The Bromeliaceae are native and Asteridae. widespread in the New World Tropics. There are two carnivorous The molecular phylogeny is startlingly different in some cases. species in Brocchinia, which is terrestrial and also limited to the The Sarraceniaceae are now placed with the Roridulaceae near the Guiana Highlands, and is an epiphyte in a group with much of the traditional Dilleniidae. While widespread from the Caribbean to the Amazon Basin (McPherson keeping the Droseraceae, Drosophyllaceae, and Nepenthaceae 2007, p. 4, 22, 40, 61). together, the molecular classification removes these to the Because Sarraceniaceae and Nepenthaceae are particularly Caryophyllales (expanded traditional subclass Carophyllidae) amenable to baraminic analysis, we limited our study to these along with Dioncophyllaceae near the Polygonaceae, a two families. They have been cultivated as a plant curiosity relationship previously never conceived. While still in the for many years, and enthusiasts have attempted many crosses, Rosidae, the Cephalotaceae are placed in the Oxalidales near the allowing us to use the hybridization criterion for membership in

JCTS B: Life Sciences www.coresci.org/jcts Volume 6:71 Table 1. Reports of Sarracenia hybrids.

rubra purpurea psittacina oreophylla minor leucophylla flava alata M, P M, P P P P P P flava M, P P P P P M. P leucophylla M, P P M, P P M minor M, P P M, P P oreophylla P P P psittacina M, P M purpurea M, P

M = MacPherson (2007) P = Pietropaolo & Pietropaolo (1986) a monobaramin (Marsh 1947, Scherer 1993, Wood et al. 2003). matK sequence (AF315866) as a query. These three outgroup We therefore surveyed reports of interspecific hybridization from sequences came from guineensis (GQ470537), published records (Clarke 1997, 2001; Clarke and Lee 2004; peltatum (AF315940), and Achatocarpus D’Amato 1998; McPherson 2007; Pietropaolo and Pietropaolo praecox (AY514845). Ancistrocladus and Triphyophyllum have 1986; Steiner 2002; Wistuba et al. 2002) and catalogues of been used as outgroups in previous phylogenetic analyses of dealers (www.flytraps.com, www.exoticaplants. Nepenthes (Meimberg et al. 2001). We aligned the sequences in com.au, www.leilaninepenthes.com, www.alohanepenthes.com, MEGA5, resulting in an alignment containing 1752 positions for www.wistuba.com). 81 different taxa. In Wise’s (1992) original description of forensic baraminology, Since the baraminic status of species was our primary objective, he recommended that molecular similarity might be used to we elected not to construct a true phylogeny for the sequences. delineate baramins. Since then, molecular data has been used Instead, we created a neighbor-joining tree (Saitou and Nei 1987) sparingly (Robinson and Cavanaugh 1998a, 1998b), mostly using counts of total nucleotide difference in place of “distances.” to determine whether nonhybridizing species of a suspected Based on the resulting tree, the degree to which nonhybridizing monobaramin might be as similar to each other as hybridizing species fall within the genetic “range” of hybridizing species can be species of the same group. Thus, if nonhybridizing species fall assessed by evaluating on what branch of the tree nonhybridizing within the molecular range of hybridizing species, they could also species appear. be reasonably included in the monobaramin of the hybridizing species. Here, we assess the molecular similarity to evaluate the Results likely baraminic membership of species that are not reported to hybridize. Within Sarraceniaceae, all pairwise hybrid combinations among the eight species of Sarracenia are known (Table 1). Hybrids were Methods also reported from the genus Heliamphora (McPherson 2007, pp. 174ff), but since Heliamphora species are still being discovered For Sarraceniaceae, we obtained eleven chloroplast DNA and described (e.g., Fleischmann et al. 2009; McPherson and sequences of the 26S ribosomal RNA gene from GenBank. The Amoroso 2011), cultivated hybrids are less well known. Fourteen sequences came from the single Darlingtonia species (DQ017390), of the 24 Heliamphora species (Table 2) are linked by hybridization two species of Heliamphora (heterodoxa [AY796056] and minor (Figure 1), but we anticipate that hybrids of the remaining species [AY727966]), and all eight species of Sarracenia (AY942694, will be discovered or produced artificially in the future. We found AY795883, DQ017391, AY796055, DQ073470, AY950690, no records of intergeneric hybrids connecting these two genera or AY727967, AY260044). As an outgroup, we included a single the third Sarraceniaceae monotypic genus Darlingtonia. sequence from dentata, which has been found to be Our molecular analysis revealed that the genera of closely similar to members of Sarraceniaceae (Conran and Dowd Sarraceniaceae are well-defined and separated (Figure 2). The 1993; Bayer et al. 1996; Neyland and Merchant 2006). We average number of single nucleotide differences (SNDs) between aligned the sequences in MEGA5 (Tamura et al. 2011), resulting Darlingtonia and Heliamphora was 42.5, between Darlingtonia in an alignment containing 870 positions for 12 different taxa. and Sarracenia 46.3, and between Sarracenia and Heliamphora For Nepenthes, we obtained 78 chloroplast DNA sequences of 30.9. SNDs within Sarraceniaceae as a whole averaged 19.3, and the trnK and maturase K (matK) genes and intergenic sequences between Sarraceniaceae and the outgroup Roridula, 55.3. from GenBank (see Appendix 1 for accession codes). We Within Nepenthaceae, species discovery continues rapidly (e.g., supplemented this set with three of the most similar non-Nepenthes Lee et al. 2006; Mey 2009; Robinson et al. 2009), and there have sequences identified in a BLAST search using theN. adnata trnK/ been some debates over specific status for particular forms (e.g.,

JCTS B: Life Sciences www.coresci.org/jcts Volume 6:72 Table 2. Heliamphora species.

1. H. arenicola 9. H. glabra 17. H. nutans 2. H. ceracea 10. H. heterodoxa 18. H. parva 3. H. chimantensis 11. H. hispida 19. H. pulchella 4. H. ciliata 12. H. huberi 20. H. purpurascens 5. H. collina 13. H. ionasi 21. H. sarracenioides 6. H. elongata 14. H. macdonaldae 22. H. tatei 7. H. exappendiculata 15. H. minor 23. H. uncinata 8. H. folliculata 16. H. neblinae

Figure 1. Hybridization network for Heliamphora species. For code to species, see Table 2.

Nepenthes murudensis). With that in mind, we here recognize Nepenthes growers. Additional hybrids will continue to be made 138 Nepenthes species (Table 3), an increase of nearly 40 since as newly discovered species become more widely available to Kurata’s (2002) enumeration. Since the goal of our analysis is to growers. In our current list, hybridizing species cross with a determine the baramin to which the Nepenthes species belong, median of three other species, but five species are reported to our list should not be considered an authoritative treatment of hybridize with more than 25 other species: N. ventricosa (38 Nepenthes species. crosses), N. maxima (32 crosses), N. veitchii (31 crosses), N. In our examination of we found records of 316 interspecific truncata (28 crosses), and N. thorelii (26 crosses) (see also Figure hybrids from published literature (Clarke 1997, 2001; Wistuba et 3). These 316 hybrids connect 95 of the ~130 Nepenthes species al. 2007; Phillipps et al. 2008; McPherson 2009; Catalano 2010; into a single monobaramin (Figure 4). McPherson and Amoroso 2011) and online Nepenthes growers’ Our molecular analysis of Nepenthes included seven species catalogues (Appendix 2). This is by no means an exhaustive list that were not part of the large, hybridizing group: N. lavicola, of Nepenthes hybrids. Undoubtedly, additional hybrids could N. treubiana, N. danseri, N. murudensis, N. ephippiata, N. be found in more obscure publications or from discussions with masoalensis, and N. pervillei. The neighbor-joining tree revealed

JCTS B: Life Sciences www.coresci.org/jcts Volume 6:73 Figure 2. Neighbor-joining tree for 26S ribosomal Figure 3. Number of hybrids reported for the most sequences derived from Sarraceniaceae species and the frequent hybridizers in Nepenthes. outgroup Roridula. a high degree of simlarity among Nepenthes species (Figure 5), Discussion as expected from previous studies (Meimberg et al. 2001). The average number of SNDs for Nepenthes-Nepenthes comparisons Based on the hybridization results presented here, each genus was 13.9. In contrast, the Nepenthes-outgroup comparisons of Sarraceniaceae can be readily identified as a monobaramin, averaged 169.5 SNDs. Two branches of Nepenthes species can even though they cannot at present be combined into a single be distinguished on the neighbor-joining tree. One group consists monobaramin by either hybridization or DNA similarity. The of N. madagascariensis, N. masoalensis, N. distillatoria, and N. case of Nepenthes is slightly more complicated. Although pervillei. The remaining Nepenthes species appear in the larger 69% of Nepenthes species (95 of 138) can be joined in a single group. monobaramin on the basis of hybridization alone, evidence is Five of the nonhybridizing species appear within the larger lacking that could combine the remaining 31% of species to the group of Nepenthes species. In particular, the sequence of N. monobaramin. Based on our molecular results, at least six (and lavicola is identical to that of the hybridizing species N. rajah. probably seven) more species can be joined to the monobaramin, The sequence of N. ephippiata differs from the sequence of the making it 73% of Nepenthes species. Given the general molecular hybridizing N. clipeata by only four nucleotides. The sequence and morphological similarity of Nepenthes species, it seems quite of N. murudensis differs from the sequence of the hybridizing likely that the remaining species will prove to be members of the N. tentacula by only three nucleotides. The sequence of N. Nepenthes monobaramin as well. treubiana differs by only six nucleotides from the sequence of the If pitcher plants originated from noncarnivorous ancestors, we hybridizing N. neoguineensis. The sequence of N. danseri differs might expect to see some variation in the carnivory habit, but by only seven nucleotides from the sequence of N. ventricosa, no such variation is observed. In both families, the carnivorous which is known to hybridize with 38 different Nepenthes species. habit is found in all species. Nevertheless, commensal and Thus, the sequences of all five of these nonhybridizing species are possibly mutualistic relationships within the pitcher exist as more similar than average (13.9 SNDs) to at least one sequence well. pitchers serves as a host to the from a hybridizing species. commensal pitcher-plant midges (Metriocnemis knabi) and The remaining two nonhybridizing species in the neighbor- mosquitoes (Wyeomyia smithii) (Heard 1994), and similar midges joining tree appear in the smaller Nepenthes group. The and mosquitoes have been reported from Heliamphora pitchers sequence of N. masoalensis differs from that of hybridizing N. (Barrera et al. 1989). Three species of nitrogen-fixing bacteria madagascariensis by only one nucleotide. The sequence of N. have also been found living in Sarracenia purpurea (Prankevicius pervillei was by far the most divergent of all nonhybridizing and Cameron 1991). Further, the production of photosynthetic Nepenthes sequences, differing an average of 25.2 SNDs from leaves preferentially to pitchers is known to be correlated with all other Nepenthes sequences tested. It was most similar to exogenous nitrogen availability in Sarracenia purpurea (Ellison sequences from N. madagascariensis and N. danseri, each of and Gotelli 2002). which differed from the N. pervillei sequence by 19 SNDs. Among the Nepenthes, there have been more fascinating reports of mutualisms and potential mutualisms. For example, tree frogs have been observed in the pitchers of Nepenthes mirabilis in China (Hua and Kuizheng 2004), and a microhylid frog is known

JCTS B: Life Sciences www.coresci.org/jcts Volume 6:74 Table 3. Nepenthes species

1. N. adnata 47. N. gymnamphora 93. N. palawanensis 2. N. alata 48. N. hamata 94. N. paniculata 3. N. alba 49. N. hamiguitanensis 95. N. papuana 4. N. albomarginata 50. N. hirsuta 96. N. peltata 5. N. ampullaria 51. N. hispida 97. N. pervillei 6. N. andamana 52. N. holdenii 98. N. petiolata 7. N. angasanensis 53. N. hurrelliana 99. N. philippinensis 8. N. appendiculata 54. N. inermis 100. N. pilosa 9. N. argentii 55. N. insignis 101. N. pitopangii 10. N. aristolochioides 56. N. izumiae 102. N. platychila 11. N. attenboroughii 57. N. jacquelineae 103. N. pulchra 12. N. baramensis 58. N. jamban 104. N. rafflesiana 13. N. beccariana 59. N. kampotiana 105. N. rajah 14. N. bellii 60. N. kerrii 106. N. ramispina 15. N. benstonei 61. N. khasiana 107. N. reinwardtiana 16. N. bicalcarata 62. N. klossii 108. N. rhombicaulis 17. N. bokorensis 63. N. kongkandana 109. N. rigidifolia 18. N. bongso 64. N. lamii 110. N. robcantleyi 19. N. boschiana 65. N. lavicola 111. N. rowanae 20. N. burbidgeae 66. N. leonardoi 112. N. sanguinea 21. N. burkei 67. N. lingulata 113. N. saranganiensis 22. N. campanulata 68. N. longifolia 114. N. sibuyanensis 23. N. ceciliae 69. N. lowii 115. N. singalana 24. N. chang 70. N. macfarlanei 116. N. smilesii 25. N. chaniana 71. N. macrophylla 117. N. spathulata 26. N. clipeata 72. N. macrovulgaris 118. N. spectabilis 27. N. copelandii 73. N. madagascariensis 119. N. stenophylla 28. N. danseri 74. N. mantalingajanensis 120. N. sumatrana 29. N. deaniana 75. N. mapuluensis 121. N. suratensis 30. N. densiflora 76. N. masoalensis 122. N. surigaoensis 31. N. diatas 77. N. maxima 123. N. talangensis 32. N. distillatoria 78. N. merrilliana 124. N. tenax 33. N. dubia 79. N. micramphora 125. N. tentaculata 34. N. edwardsiana 80. N. mikei 126. N. tenuis 35. N. ephippiata 81. N. mindanaoensis 127. N. thai 36. N. epiphytica 82. N. mira 128. N. thorelii 37. N. eustachya 83. N. mirabilis 129. N. tobaica 38. N. eymae 84. N. mollis 130. N. tomoriana 39. N. faizaliana 85. N. monticola 131. N. treubiana 40. N. flava 86. N. muluensis 132. N. truncata 41. N. fusca 87. N. murudensis 133. N. undulatifolia 42. N. gantungensis 88. N. naga 134. N. veitchii 43. N. glabrata 89. N. neoguineensis 135. N. ventricosa 44. N. glandulifera 90. N. nigra 136. N. vieillardii 45. N. gracilis 91. N. northiana 137. N. villosa 46. N. gracillima 92. N. ovata 138. N. vogelii

JCTS B: Life Sciences www.coresci.org/jcts Volume 6:75 Figure 4. Hybridization network for Nepenthes species. For code to species, see Table 3.

to breed in the pitchers of Nepenthes ampullaria in Borneo (Das each family survived the Flood, or at least one species in each and Haas 2010). In 2009, Clarke et al. reported a novel mutualism monobaramin, if the three monobaramins of Sarraceniaceae also in which tree shrews (Tupaia montana) feed on exudates produced should prove to be apobaramins. The present data, however, by Nepenthes lowii and defecate into their pitchers. The nitrogen are insufficient to address the apobaraminic status of these two subsequently acquired from the feces accounted for more than half families or their genera. of the nitrogen acquired by N. lowii plants. Mutualisms between Even though the Cephalotaceae and carnivorous Bromeliaceae defecating tree shrews and two additional Nepenthes species have were not included in this study, certain observations are relevant. also been reported (Chin et al. 2010, see also Clarke et al. 2010). The morphological isolation and lack of apparent affinity to Most recently, Grafe et al. (2011) reported a mutualism between other families suggest the Cephalotaceae is an apobaramin Hardwicke’s woolly bats (Kerivoula hardwickii hardwickii) and consisting of a single species, which by default would also make Nepenthes rafflesiana var. elongata, where the plants gained as it a monobaramin, and thus, a holobaramin. Thus the conclusion much as a third of their nitrogen from the feces of bats taking of pre-Flood design would also apply to this family. Was the refuge in the pitchers but above the digestive fluid. These fecal- Cephalotaceae more speciose before the Flood, and why has there based mutualisms are certainly suggestive of a possible non- been a lack of diversification after the Flood? These are questions carnivorous role for pitchers in a pre-Fall world. for future research. Therefore, we propose that in the Sarraceniaceae and On the other hand the three species of Bromeliaceae suggest a Nepenthaceae, the origin of the carnivorous morphology may different picture. Since these species occur in two genera with have resulted from creation week events. That is, these plants many non-carnivorous species each, we would propose that these were designed for symbiotic relationships to supply nitrogen in are examples of mediated design appearing concurrently with post- some unusual nitrogen deficient pre-Flood habitat or habitats. Flood diversification, at least within the two genera. However, However, the habitats in the present world where these species the morphology of these pitchers are starkly different from those thrive probably were not present or as diverse in the pre-Flood of the Sarraceniaceae, Nepenthaceae, and Cephalotaceae. Most world. This leads us to speculate that at least one species in Bromeliaceae are predisposed to form water-holding tanks from

JCTS B: Life Sciences www.coresci.org/jcts Volume 6:76 Figure 5. Neighbor-joining tree for trnK and matK sequences derived from Nepenthes and three outgroup species. Nonhybridizing Nepenthes species are indicated in red.

the splayed but tightly overlapping leaf bases. So we view Bayer, R.J., L. Hufford, D.E. Soltis. 1996. Phylogenetic carnivory in this case as a likely modification of a creation- relationships in Sarraceniaceae based on rbcL and ITS event structural distinction of bromeliads that lacks evidence of sequences. Systematic Botany 21(2):121-134. precision symbiosis. Catalano, M. 2010. Nepenthes della Thailandia. Self-published, Despite these fascinating mutualisms in the Sarraceniaceae and Prague. Nepenthaceae, we must note that the carnivorous habit of most Chin, L., J.A. Moran, and C. Clarke. 2010. Trap geometry in three pitcher plants is well-characterized and highly sophisticated. giant montane species from Borneo is a function Beyond just the secretion of digestive enzymes in the pitcher of tree shrew body size. New Phytologist 186:461-470. fluid, pitcher plants are known to lure prey with floral scent Clarke, C.M. 1997. Nepenthes of Borneo. Natural History mimicry (Di Giusto et al. 2010) and ultraviolet markings (Joel Publications, Kota Kinabalu, Malaysia. et al. 1985). Further, pitcher plants get a large fraction of their Clarke, C.M. 2001. Nepenthes of Sumatra and Peninsular nitrogen from insect prey (e.g., Schulze et al. 1997). The origin Malaysia. Natural History Publications, Kota Kinabalu, of these sophisticated prey capture systems goes far beyond just Malaysia. a slight modification from an otherwise benign condition. These Clarke, C.M., U. Bauer, C.C. Lee, A.A. Tuen, K. Rembold, and attributes suggest some sort of intentional design. J.A. Moran. 2009. Tree shrew lavatories: a novel nitrogen sequestration strategy in a tropical pitcher plant. Biology References Letters 5:632-635. Clark, C., J.A. Moran, and L. Chin. 2010. Mutualism between Barrera, R., D. Fish, and C.E. Machado-Allison. 1989. Ecological tree shrews and pitcher plants: perspectives and avenues for patterns of aquatic insect communities in two Heliamphora future research. Plant Signaling & Behavior 5(10):1187-1189. pitcher-plant species of the Venezuelan highlands. Ecotropicos Clarke, C.M. and C.C. Lee. 2004. Pitcher Plants of Sarawak. 2(1):31-44. Natural History Publications, Kota Kinabalu, Malaysia.

JCTS B: Life Sciences www.coresci.org/jcts Volume 6:77 Conran, J.G. and J.M. Dowd. 1993. The phylogenetic relationships Molecular phylogeny of Nepenthaceae based on cladistic of and Roridula (Byblidaceae-Roridulaceae) inferred analysis of plastic trnK intron sequence data. Plant Biology from partial 18S ribosomal RNA sequences. Plant Systematics 3(2):164-175. and Evolution 188:73-86. Mey, F.S. 2009. Nepenthes bokorensis, a new species of Cronquist, A. 1981. An integrated system of classification of Nepenthaceae from Cambodia. Carniflora Australis 7(1):6-15. flowering plants. Columbia University Press, , NY. Neyland, R and M. Merchant. 2006. Systematic relationships D’Amato, P. 1998. The Savage Garden: Cultivating Carnivorous of Sarraceniaceae inferred from nuclear ribosomal DNA Plants. Ten Speed Press, Berkeley, CA. sequences. Madroño 53(3):223-232. Das, I. and A. Haas. 2010. New species of Microhyla from Phillipps, A., A. Lamb, and C. Lee. 2008. Pitcher Plants of Sarawak: Old World’s smallest frogs crawl out of miniature Borneo. 2nd ed. Natural History Publications, Borneo. pitcher plants on Borneo (Amphibia: Anura: Microhylidae). Pietropaolo, J. and P. Pietropaolo. 1986. Carnivorous Plants of Zootaxa 2571:37-52. the World. Timber Press, Portland, OR. Di Giusto, B., J.-M. Bessière, M. Guéroult, L.B.L. Lim, D.J. Prankevicius, A.B. and D.M. Cameron. 1991. Bacterial dinitrogen Marshall, M. Hossaert-McKey, and L. Gaume. Flower-scent fixation in the leaf of the northern pitcherSarracenia plant( mimicry masks a deadly trap in the carnivorous plant Nepenthes purpurea). Canadian Journal of Botany 69:2296-2298. rafflesiana. Journal of Ecology 98:845-856. Robinson, A.S., A.S. Fleischmann, S.R. McPherson, V.B. Ellison, A.M. 2006. Nutrient limitation and stoichiometry of Heinrich, E.P. Gironella, and C.Q. Peña. 2009. A spectacular carnivorous plants. Plant Biology 8:740-747. new species of Nepenthes L. (Nepenthaceae) pitcher plant from Ellison, A.M. and N.J. Gotelli. 2002. Nitrogen availability central Palawan, Philippines. Botanical Journal of the Linnean alters the expression of carnivory in the northern pitcher plant, Society 159:195-202. Sarracenia purpurea. Proceedings of the National Academy of Saitou, N. and M. Nei. 1987. The neighbor-joining method: a Sciences USA 99:4409-4412. new method for reconstructing phylogenetic trees. Molecular Ellison, A.M. and N.J. Gotelli. 2009. Energetics and the evolution Biology and Evolution 4:406-425. of carnivorous plants - Darwin’s ‘most wonderful plants in the Schulze, W., E.D. Schulze, J.S. Pate, and A.N. Gillison. 1997. world.’ Journal of Experimental Botany 60:19-42. The nitrogen supply from and insects during growth of the Grafe, T.U., C.R. Schöner, G. Kerth, A. Junaidi, and M.G. pitcher plants Nepenthes mirabilis, Cephalotus follicularis, and Schöner. 2011. A novel resource-service mutualism between Darlingtonia californica. Oecologia 112:464-471. bats and pitcher plants. Biology Letters 7:436-439. Soltis, D.E., P.S. Soltis, P.K. Endress and M.W. Chase. 2005. Heard, S.B. 1994. Pitcher-plant midges and mosquitoes: a Phylogeny and Evolution of Angiosperms. Sinauer Associates, processing chain commensalism. Ecology 75(6):1647-1660. Sunderland, MA. Hua, Y. and L. Kuizheng 2004. The special relationship between Steiner, H. 2002. Borneo: Its Mountains and Lowlands with their Nepenthes and tree frogs. Carnivorous Plant Newsletter Pitcher Plants. Toihaan Publishing Company, Kota Kinabalu, 33(1):23-24. Malaysia. Joel, D.M., B.E. Juniper, and A. Dafni. 1985. Ultraviolet patterns Takhtajan, A. L. 1980. Outline of the classification of flowering in the traps of carnivorous plants. New Phytologist 101:585- plants (Magnoliophyta). Botanical Review 46: 225-359. 593. Tamura, K., D. Peterson, N. Peterson, G. Stecher, M. Nei, and Kurata, S. 2002. Revision trial in recent enumeration of Nepenthes S. Kumar. 2011. MEGA5: Molecular Evolutionary Genetics species. Proceedings of the 4th International Carnivorous Plant Analysis using maximum likelihood, evolutionary distance, Conference. International Carnivorous Plant Society, Tokyo, and maximum parsimony methods. Molecular Biology and pp. 111-116. Evolution doi: 10.1093/molbev/msr121. Lee, C.C., Hernawati, and P. Akhriadi. 2006. Two new species Thorne, R. F. 1976. A phylogenetic classification of the of Nepenthes (Nepenthaceae) from North Sumatra. Blumea Angiospermae. Evoutionary Biology 9: 35-106. 51:561-568. Wistuba, A., T. Carow, and P. Harbarth. 2002. Heliamphora McPherson, S. 2007. Pitcher Plants of the . McDonald chimantensis, a new species of Heliamphora (Sarraceniaceae) & Woodward Publ., Blacksburg, VA. from the ‘Macizo de Chimanta’ in the south of Venezuela. McPherson, S. 2009. Pitcher Plants of the Old World. Redfern Carnivorous Plant Newsletter 31(3):78-82. Natural History Productions. Wistuba, A., J. Nerz, and A. Fleischmann. 2007. Nepenthes McPherson, S. and V.B. Amoroso. 2011. Field Guide to the flava, a new species of Nepenthaceae from the northern part of Pitcher Plants of the Philippines. Redfern Natural History Sumatra. Blumea 52:159-163 Productions. Wood, T. C., K. P Wise, R. W Sanders, and N. Doran. 2003. Meimberg, H., A. Wistuba, P. Dittrich, and G. Heubl. 2001. A refined baramin concept. Occasional Papers of the BSG 3: 1-14.

JCTS B: Life Sciences www.coresci.org/jcts Volume 6:78 Appendix 1. Genbank accession codes for Nepenthes sequences used in this study.

N. adnata AF315866 N. madagascariensis AF315883 N. alata AF315891 N. mapuluensis AF315918 N. albomarginata AF315908 N. masoalensis AF315884 N. ampullaria AF315888 N. maxima AF315913 N. aristolochioides AF315900 N. merrilliana AF315912 N. bellii AF315916 N. mikei AF315911 N. bicalcarata DQ007089 N. mira DQ007085 N. bongso AF315865 N. mirabilis AF315920 N. boschiana AF315903 N. muluensis AF315933 N. burbidgeae AF315921 N. murudensis DQ007084 N. burkei DQ840247 N. neoguineensis AF315896 N. clipeata AF315877 N. northiana AF315901 N. danseri DQ007087 N. ovata AF315873 N. densifloraAF315927 N. pervillei AF315885 N. diatas AF315915 N. petiolata AF315902 N. distillatoria AF315886 N. pilosa AF315919 N. dubia AF315869 N. rafflesianaAF315910 N. edwardsiana DQ840248 N. rajah AF315879 N. ephippiata AF315906 N. ramispina DQ007083 N. eustachys AF315867 N. reinwardtiana AF315907 N. eymae AF315930 N. rhombicaulis AF315874 N. faizaliana AF315917 N. sanguinea AF315923 N. fusca AF315936 N. sibuyanensis DQ840246 N. glabrata AF315928 N. singalana DQ007082 N. gracilis AF315937 N. spathulata DQ007081 N. gracillima DQ007086 N. spectabilis AF315868 N. gymnamphora AF315864 N. stenophylla AF315922 N. hamata AF315914 N. sumatrana AF315872 N. hirsuta AF315889 N. talangensis AF315924 N. inermis AF315870 N. tentaculata AF315932 N. insignis AF315881 N. thorelii AF315890 N. khasiana AF315887 N. tobaica AF315899 N. lamii AF315905 N. tomoriana AF315898 N. lavicola AF315935 N. treubiana AF315893 N. longifolia AF315871 N. truncata AF315904 N. lowii AF315875 N. veitchii AF315895 N. macfarlanei AF315894 N. ventricosa AF315892 N. macrophylla AF315931 N. vieillardii AF315897 N. macrovulgaris AF315934 N. villosa AF315925

JCTS B: Life Sciences www.coresci.org/jcts Volume 6:79 Appendix 2. Published Nepenthes hybrids. See Table 3 for species codes.

1 × 37 16 × 83 45 × 83 77 × 10 107 × 129 128 × 5 2 × 10 16 × 91 45 × 91 77 × 19 108 × 92 128 × 10 2 × 30 16 × 118 45 × 104 77 × 78 108 × 118 128 × 30 2 × 61 18 × 77 45 × 106 77 × 82 108 × 129 128 × 32 2 × 77 18 × 105 45 × 120 77 × 91 109 × 118 128 × 41 2 × 78 18 × 115 45 × 128 77 × 112 112 × 22 128 × 45 2 × 83 18 × 132 45 × 134 77 × 118 112 × 70 128 × 61 2 × 103 20 × 34 46 × 2 77 × 119 112 × 78 128 × 107 2 × 114 20 × 41 46 × 135 77 × 123 112 × 114 128 × 111 2 × 118 20 × 134 47 × 18 77 × 128 112 × 132 128 × 118 2 × 119 21 × 135 47 × 19 77 × 130 112 × 134 128 × 129 2 × 129 22 × 77 47 × 56 77 × 132 114 × 5 128 × 132 2 × 132 22 × 134 47 × 115 77 × 134 114 × 41 128 × 134 2 × 134 23 × 103 50 × 16 78 × 55 114 × 77 128 × 135 2 × 135 25 × 134 50 × 41 78 × 132 114 × 123 129 × 4 4 × 16 26 × 21 50 × 104 80 × 47 114 × 132 129 × 69 4 × 45 26 × 34 50 × 112 81 × 132 114 × 135 129 × 92 4 × 77 26 × 38 50 × 118 81 × 134 117 × 2 129 × 118 4 × 91 26 × 77 50 × 119 83 × 104 117 × 5 129 × 128 4 × 107 26 × 104 50 × 134 83 × 119 117 × 10 129 × 132 4 × 128 26 × 107 50 × 135 83 × 120 117 × 16 129 × 134 4 × 134 26 × 128 51 × 107 83 × 128 117 × 18 132 × 22 5 × 14 26 × 134 53 × 69 83 × 130 117 × 19 132 × 30 5 × 16 26 × 135 54 × 18 83 × 134 117 × 22 132 × 48 5 × 45 31 × 80 54 × 115 86 × 69 117 × 30 132 × 72 5 × 50 32 × 45 54 × 123 86 × 125 117 × 41 132 × 82 5 × 78 32 × 134 54 × 135 89 × 5 117 × 69 132 × 119 5 × 83 32 × 135 56 × 132 89 × 77 117 × 77 132 × 134 5 × 91 33 × 47 57 × 56 92 × 118 117 × 104 134 × 16 5 × 104 33 × 115 59 × 77 93 × 99 117 × 118 134 × 19 5 × 111 34 × 137 59 × 135 98 × 132 117 × 119 134 × 91 5 × 129 37 × 68 61 × 106 100 × 134 117 × 122 134 × 102 5 × 135 37 × 77 61 × 112 104 × 16 117 × 125 135 × 4 6 × 83 37 × 120 61 × 114 104 × 77 117 × 128 135 × 10 7 × 30 37 × 128 61 × 132 104 × 78 117 × 132 135 × 18 10 × 61 37 × 134 61 × 135 104 × 111 117 × 134 135 × 22 10 × 115 38 × 41 62 × 77 104 × 114 117 × 135 135 × 30 10 × 132 38 × 77 63 × 83 104 × 118 118 × 45 135 × 55 13 × 118 38 × 118 64 × 136 104 × 119 118 × 68 135 × 72 14 × 81 38 × 134 69 × 10 104 × 128 118 × 91 135 × 75 14 × 114 39 × 134 69 × 21 104 × 129 118 × 106 135 × 77 14 × 117 40 × 92 69 × 22 104 × 132 118 × 123 135 × 80 14 × 118 40 × 108 69 × 71 104 × 134 118 × 132 135 × 81 14 × 119 41 × 4 69 × 100 104 × 135 118 × 134 135 × 83 14 × 128 41 × 77 69 × 119 105 × 137 118 × 135 135 × 92 14 × 132 41 × 107 69 × 132 106 × 2 119 × 128 135 × 106 14 × 134 41 × 112 69 × 134 106 × 70 119 × 134 135 × 112 14 × 135 41 × 118 69 × 135 106 × 112 123 × 82 135 × 119 15 × 83 41 × 134 69 × 137 106 × 132 123 × 129 135 × 123 16 × 5 43 × 77 70 × 41 107 × 72 123 × 132 135 × 125 16 × 22 43 × 125 70 × 114 107 × 77 124 × 83 135 × 132 16 × 45 45 × 61 70 × 135 107 × 115 124 × 111 16 × 77 45 × 77 73 × 135 107 × 119 128 × 2

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