Abstract
We classify Nepenthes species into 12 functional pitcher types, based on combinations of traits that appear to comprise different syndromes for capturing nutrients, usually from animals. For nine of these types the trapping syndromes are already documented, six targeting live animals (hence carnivorous), and three targeting other nutrient sources (non-carnivorous). Yet, for three pitcher types here is no previous documentation of the syndrome and we do not yet know what sources of nutrients are being targeted. Mapping all these pitcher types on the latest, near comprehensive species-level phylogenomic tree of Nepenthes (Murphy et al. 2019) shows that apart from the ancestral pitcher type 1, most of the remaining pitcher types have evolved independently, in different parts of the phylogenetic tree, usually in several different places. Each of the 12 pitcher types is characterised morphologically and illustrated, its trapping syndrome discussed, and example species are given. An identification key to the 12 pitcher types is presented. The possibility of additional pitcher types being present is discussed.
Introduction
The pitchers of Nepenthes come in many shapes, sizes, and colours. Within a single individual these features vary, sometimes dramatically, as the plant matures. One of the most characteristic features of these plants is the dimorphy of pitchers in most species. These are distinguished as upper and lower pitchers, occasionally with distinct intermediate forms. The switch from one form to another is associated with the onset of elongate stems and flowering (Jebb, 1991). It is the upper pitchers (or in those few species that do not form upper pitchers, the intermediate or lower pitchers) that usually characterise a species, possessing the features that enable it to be identified and separated from other species.
Historically the presumption of insectivory meant that little attention was paid to the subtlety of pitcher shape and form in this regard. It is now recognised that different species of Nepenthes can obtain their nutrients from several different, mainly animal, sources, using a diversity of mechanisms and features or characters present in their pitchers, that therefore appear to be “functional traits” (i.e. Bauer et al. 2012a).
Based on such traits the pitchers of the known species of Nepenthes are classified here into 12 different functional types. Nine of these types correspond to different, more-or-less well-documented mechanisms for obtaining nutrients. But, for three of these types we can only conjecture what mechanisms might be used and what sources of nutrients might be targeted.
Two of the 12 types have only a single documented species, such as type 10, represented only by N. albomarginata Lobb ex Lindl. At present this is the only species known to specialise exclusively in trapping termites, which it entices by means of the properties of the white hairy band on the outside of the pitcher below the peristome. No other species has such a band. However, ten of the 12 types have two to many species, and with the exception of the “ancestral” pitcher type 1, they are scattered as individuals or in clusters, through the evolutionary tree of Nepenthes (Murphy et al. 2019). It is therefore clear that nine of the 12 types documented here have each arisen independently more than once.
Materials and Methods
Following a review of the literature on Nepenthes trapping syndromes, the species of the genus were classified into nine discrete morphological groups (each linked to a different syndrome) based on the functional traits present in their latest stage, usually upper, pitchers. Those species not falling into these nine groups fell into three further distinct morphological groups (pitcher types 4, 6 and 12) which were also characterised. An identification key to the 12 pitcher types was constructed using conventional taxonomic methods. Morphological data (e.g. pitcher shape and proportions) were taken from Jebb & Cheek (1997) and Cheek and Jebb (2001) and for species published subsequently, the protologues (e.g. Cheek & Jebb 2013a-h). Presence or absence of a “conductive” or waxy zone in the pitchers, not usually recorded in taxonomic literature, was obtained by study of reference herbarium specimens at the Kew Herbarium (K), and in some cases where specimens were not available, by reference to photographs available in McPherson (2009). Waxy zones have an opaque, dull, off-white appearance, while non-waxy surfaces have a glossy appearance. Presence or absence of visco-elastic versus watery pitcher fluid, also not usually recorded in taxonomic literature, was obtained from observation of late stage pitchers from live plants of 30 representative species in the Tropical Nursery of the Royal Botanic Gardens, Kew, U.K. in Nov. 2019, and (one species) at the National Botanic Gardens, Ireland (Table 1), also from the literature cited in the accounts of pitcher types 1-12 below. Visco-elastic fluid was detected in the species listed in table 1, by inserting two fingers into the pitcher fluid, withdrawing them from the fluid and then separating the two fingers. In visco-elastic species a filament is developed between the fingers, but is absent in non-visco-elastic species. The figures representing the 12 pitcher types are all drawn by Matthew Jebb from photos of live plants identified using Cheek & Jebb (2001).
Results
A fundamental division between pitcher types is seen on the inner surface of the pitcher, between “waxy” (types 1, 7, 8, (9), 10) and “non-waxy” (types 2-6, (9), 11 & 12) species. It was Macfarlane (1908: 20) who first wrote in detail about waxy versus non-waxy zones and about the variation from one species to another. Bauer et al. (2012a) later documented this fundamental division in more detail (see below). The fundamental type of Nepenthes pitchers, type 1 of this classification, is seen in the earliest diverging, ancestral species such as Nepenthes pervillei Bl. and Nepenthes distillatoria L.f., but type 1 pitchers also predominate in Sect. Montanae Danser of the mountains of Sumatra and the Malay Peninsula e.g. N. sanguinea Lindl., and Sect. Pyrophytae Cheek & Jebb of Indo-china, e.g N. smilesii Hemsl. Most species of Sect. Alatae Cheek & Jebb (in the wide sense), from Luzon to Mindanao, also have type 1 pitchers.
Key to functional pitcher types of Nepenthes (based on the latest stage pitchers developed)
Pitchers with a pale, matt, waxy (conductive) zone on the upper inner surface below the peristome, separated the from the glossy (detentive) zone at the base of the pitcher by an external visible raised line, the ‘hip’; liquid usually non-viscous ………………………………2
Pitchers entirely glossy on the inner surface, lacking matt, waxy zone, or with only a vestigial triangular remnant below the insertion of the lid, hip absent; liquid often (types 2, 3, 12 and N. aristolochioides) viscous …………………………………………………………………..6
Pitchers with a white hair band on the outer surface below the peristome
………………………………………………………………….. Type 10, Termite Trap
Pitchers lacking a white hair band …………………………………………………………………..3
Lower surface of pitcher lid with either wax scales or hairs in the central portion
………………………………………………………………….. Type 7. Flick of the lid
Lower surface of the lid lacking wax scales or hairs in the central portion………………………………4
Pitchers highly elongated, length: breadth ratio c. 10: 1 frequently acting as a day roost for bats ………………………………………………………………….. Type 8. Bat-roost
Pitchers not highly elongated, length: breadth ratio <8: 1 not (or infrequently) acting as a roost for bats ………………………………………………………………….. 5
Pitchers with mouth lateral, vertical; apical windowed dome ………………………………Type 9. Light-trap
Pitchers with mouth apical, horizontal, lacking an apical dome ………………………………Type 1. Generalist
Pitchers narrowly funnel-shaped …………………………………………………………………..Type 2. Narrow-funnel
Pitchers ellipsoid, broadly cylindrical, domed, or broadly funnel-shaped ………………………………7
Pitchers often reclining, ellipsoid, usually massive and subwoody, diameter of mouth c. 10 cm; lid held erect of reflexed …………………………………………………………………..Type 5. Tree shrew lavatory
Pitchers erect not reclining, not ellipsoid, but broadly cylindrical, domed or broadly funnel-shaped ………………………………………………………………….. 8
Pitchers broadly funnel-shaped, length: breadth ratio 1 to 1.5:1; usually 3-5 cm long, the upper part cup or bowl-shaped, wider than long, the lower part narrowly cylindrical ………………………………9
Pitchers broadly cylindrical or domed …………………………………………………………………..10
Pitchers lacking a very broad (to 3.5 cm wide), peristome absent or <1 cm wide
…………………………………………………………………………………………… ………………Type 3. Flypaper
Pitchers with a very broad (to 3.5 cm wide), flat peristome ………………………………Type 12. Flat Lip
Pitchers with mouth lateral, vertical, apical windowed dome ………………………………Type 9. Light-trap
Pitchers with mouth apical, horizontal or inclined, lacking a windowed dome ……………………………… 11A
Lid held on a well-developed vertical column; outer surface of pitcher often with long, early caducous hairs …………………………………………………………………..Type 6. Globose-hairy
Lid lacking a distinct column; peristome ridges not wing-like, hairs if present stellate ………………………………12
Pitchers never in carpets, with lid length: breadth 1.5-2: 1, held over the mouth, with nectar glands on the lower surface …………………………………………………………………..Type 4. Stout Cylinder
Pitchers usually in carpets on the ground, with lid narrowly oblong length: breadth ratio c. 4:1, reflexed, not held over mouth, lacking nectar glands ………………………………Type 11. Pitfall
Type 1 species seem to derive their nutrition, so far as is known, by trapping a wide range of insects, as documented in detail by Jebb (1991) for Nepenthes mirabilis in Papua New Guinea. However, the number of prey individuals is dominated by ants: >80 % of prey in both upper and lower pitchers. More recent analysis of trapping syndromes in six species of Nepenthes in Brunei concluded that cylindrical pitchers with waxy walls (type 1) are particularly efficient at trapping and retaining ants, compared with other syndromes (Gaume et al. 2016). Earlier, Bonhomme et al. (2011) concluded that “wax only appears to be efficient for ants”.
Notably, we have not detected viscid fluid in the non-waxy pitcher types 4, 5, 6 and 11). Moran et al. (2012) also record visco-elastic fluid occurring in some waxy species, such as N. tobaica Danser, yet our checks of several accessions of this species showed it to be non-visco-elastic (Cheek pers. obs 2019). Gaume et al. (2016) record viscid fluid in N.hemsleyana Macfarl. (which we confirmed) which is also waxy, and we confirmed this from observations of live plants cultivated at Kew.
Species with type 2, non-waxy, narrowly funnel-shaped upper pitchers, can have type 1 waxy, ovoid-cylindric lower pitchers on the same plant e.g. N.rafflesiana. A key element of this stage-dependent heteromorphy (difference in shape dependent on stage of development) explains the loss of the waxy zone. This seems to be the switch in pitcher shape. Lower pitchers are usually ovoid-cylindric (also with the tendril uncoiled, placed in front of the mouth, and with the front of the pitcher facing the stem and having a pair of fringed wings). Upper pitchers instead, are more or less funnel-shaped in type 2 (and generally also face away from the stem, and have the tendril coiled and arising at the rear of the pitcher, and the fringed wings reduced to a pair of low ridges). This change in shape seems to be important in deciding whether or not the pitcher is waxy on the inner surface, or not. While species with narrowly cylindrical or ovoid-cylindric upper pitchers retain the waxy zone, those species which have funnel-shaped pitchers (widening gradually from base to apex) lack a waxy zone, or have only a vestigial, minute waxy triangle as described above. It is as though in the funnel-shaped pitchers the development of the upper cylindrical waxy zone is “switched off”, the ovoid basal non-waxy detentive zone instead expanding to fill the gap, and directly support the peristome instead. Gaume et al. (2016) report that in N. rafflesiana not only does the shape and waxiness of the pitcher switch from lower to upper pitcher, but that lower pitchers have non-viscid fluid, while upper pitchers have viscid fluid. In addition, the upper pitchers (type 2) of this species capture more flying insects than do their type 1 lower pitchers. In a study of six different Nepenthes species co-occurring at two sites in lowland Brunei, Gaume et al. (2016) found that pitcher shape critically influences the capture of flying insects, stating that “flying insects are clearly associated with funnel-shaped pitchers”.
In contrast species with waxy, narrow, cylindrical pitchers (e.g type 1 and type 10) proved more effective in trapping ants and termites respectively.
Nepenthes madagascariensis Poir. is exceptional in that it is the only species in the basal grade of the genus that does not have type 1 pitchers. Its narrow funnel upper pitchers, typical of those in type 2, were found to trap significantly more Coleoptera, Diptera and Lepidoptera (flying insects) “than in all other Nepenthes species” (Rembold et al. 2010). However, unusually for type 2, it does not have visco-elastic but watery fluid (Cheek pers. obs.). Bonhomme et al. (2011) hypothesised that the visco-elastic fluids in Nepenthes, Drosera, and Drosophyllum (in the last two genera appearing as insect-trapping mucilage on the leaf tentacles) have a common and thus a plesiomorphic origin. However, evidently the visco-elastic liquid trait lay dormant or was not re-acquired until after the genus arrived in Borneo. The earliest branching species of Nepenthes in which it occurs is N. rafflesiana.
Visco-elastic fluid is effective at retaining small prey, such as small ants e.g. Oecophylla smaragdina which are totally unable to free their bodies from it. However, larger ants, such as Polyrhachis species, can haul themselves out, and climb up the non-waxy pitcher wall in Nepenthes rafflesiana (Gaume & Forterre 2007).
However, species with waxy zones are far more effective at retaining prey at the bottom of their walls than those species with non-waxy walls. Comparing the similar and closely related Nepenthes rafflesiana var. elongata (that is N. hemsleyana) which has waxy walls, with N. rafflesiana var. typica (that is N. rafflesiana) which is non-waxy, Gaume & di Gusto (2009) found in experiment that the former retains 73.5% of trapped individuals, while the latter only 28.5%.
Bonhomme et al. (2011) quantified visco-elasticity and wax-levels of 23 cultivated species of Nepenthes, measuring their effects on the retention rates of flies and ants placed in the lower pitchers of 12 of these species. In the 23 species they found that none of the species with high levels of waxiness was found to exhibit a very visco-elastic fluid, and vice versa. They concluded that there were two strategies: a ‘waxy’ strategy and a ‘visco-elastic’ strategy. While retention rates for ants increased with waxiness and visco-elasticity, for flies retention rates did not depend on the amount of wax but were far higher when the fluid was visco-elastic. Two thirds of the 23 species in their study were classified as visco-elastic and these species tended to be montane rather than lowland. This agrees with the observations of Collins (1980) that in montane habitats in Borneo, ants are relatively fewer in number, but flying insects relatively more abundant.
This prey-type and trapping mechanism has been recorded in the unrelated N. eymae Danser of the Regiae section in Sulawesi (Cheek & Jebb 2001: 82, McPherson 2009: 993). Two atypical species of Sect. Tentaculatae Cheek & Jebb in Sulawesi also have type 3 pitchers e.g. N.pitopangii Chi.C.Lee et al., and N. undulatifolia Nerz et al, and in New Guinea, N. paniculata Danser. Most of the type 3 pitcher species however occur with N. inermis in a group of Sumatran Montanae which were characterised in Cheek et al. (2017) as subsect. Poculae-Ovis (“the egg-cups”, due to their size and shape), so that type 3 pitchers have arisen independently in four different parts of the Nepenthes evolutionary tree.
This distinctive pitcher type was previously considered to represent a closely related, natural group of species. The term villosa group or complex was coined after the discovery of N. mira from Palawan when it was placed with N. villosa. of Kinabalu and its relatives in NE Borneo due to their morphological similarities. to these were added the minute but morphologically similar N. argentii Jebb & Cheek of Sibuyan (Cheek & Jebb 1999). The term villosa group or complex was adopted and expanded by Robinson et al. (2009) to include two newly discovered species from Palawan, and also N.peltata Sh.Kurata of Mindanao. However, the molecular phylogenetic work of Murphy et al. (2019) showed that the NE Borneo, Palawan, Sibuyan and Mindanao species referred to above fall in four separate clades so that their morphological similarity must be due to convergence. However, the source of nutrition targeted by species with this type of pitcher is not known. All species are restricted to high altitude ultramafic habitats.
Discussion
Further pitcher types are likely to be discovered in Nepenthes as more species are discovered, and as those that are known are better researched. For example, Nepenthes bicalcarata here included in pitcher type 4, differs from all other species ascribed to that pitcher type (and from all other Nepenthes) in a) its mutualism with the specialised ant Camptonopus schmitzii which it accomodates, and b) in its fang-like peristome extensions, so that it may prove to represent a further trapping syndrome.
There are few studies that compare trapping of prey by different species of Nepenthes with different pitcher types at a single site. Among the ten species in Sumatra studied by Kato et al. (1993) three species, equating to different pitcher types, were sympatric at in the submontane forests of Gunung Gadang. These were, N. inermis (type 3), N. spathulata Danser (type 2) and Nepenthes B (unidentified, probably type 1). All three had different prey assemblages. The availability of prey was thought to be largely similar among the three species because their microhabitats were largely similar. This suggested to Kato et al. that the differences between prey assemblages was due to different prey trapping patterns.
A second study by Gaume et al. (2016) in lowland heath forest of Brunei studied six species, each with a different pitcher type: N. rafflesiana, type 2; N. bicalcarata, type 4; N. gracilis, type 7; N. hemsleyana, type 8; N. albomarginata, type 10 and N. ampullaria, type 11). All six species were present at two sites. They excluded from consideration N. hemsleyana (bat droppings, type 8) and N. ampullaria (litter, type 11) since they are largely non-carnivorous. Their analysis stated that there were three main carnivorous syndromes A) The “flying insect syndrome”, characterized by funnel-shaped pitchers of large diameters, with a yellow dominant colour, an acidic viscoelastic fluid, nectar secretion, and the delivery of a sweet scent; B) The “ant syndrome”, which is less specific and is characterized primarily by nectar secretion, then by fluid acidity and, to a lesser extent, a waxy trap; and finally C) The “termite syndrome”, characterized by narrower pitchers and a shape that is closer to a cylinder, with non-viscous fluids. Termite capture is also greatly enhanced by the presence of a rim of edible trichomes or the symbiotic presence of the hunter ant, C. schmitzi. The flying insect syndrome clearly maps onto N. rafflesiana (type 2), while the termite syndrome maps onto type 10 (N.albomarginata), and to some extent also type 4 (N. bicalcarata). Ant trapping is more widely spread among the species, but linked especially with N. gracilis, although Gaume et al. appear unaware of the “Flick of the Lid” mechanism revealed by Bauer et al. (2012b). These two studies, in different geographic locations, in different habitats at different altitudes, indicate that sympatric species of Nepenthes have different prey assemblages, and different trapping mechanisms. Additional studies are needed to test whether this is always the case.
Additional cryptic pitcher traits that are likely to be important in trapping syndromes, but for which both comparative and experimental data are absent or very sparse are: a) pitcher fragrance (Di Gusto et al. 2008); b) acidity levels of pitcher fluid (eg. Gaume et al. 2016); c) enzymatic differences in the pitcher fluid (Biteau et al. 2013); d) lower lid nectar gland and appendage structures (e.g. Cheek 2015); and e) peristome ridge and teeth morphology.
Pitcher morphology might not always be related solely to nutrient trapping. Water storage as a buffer for dry periods has not been adequately researched as a potential function of pitchers.
ACKNOWLEDGEMENTS
Thanks to the past and present staff of the tropical nursery, especially Kath King, Nick Johnson, James Beattie, Rebecca Hilgenhof and Tom Pickering for maintaining the live Nepenthes collection that has been so important for our ongoing studies of Nepenthes. We thank Tim Barraclough, Magdalen College, University of Oxford for suggesting that we categorise Nepenthes species by pitcher type.