Implementation and Monitoring of Soil Bioengineering Measures at a Landslide in the Middle Mountains of Nepal

Legume tissue quality is a key factor for enhancement of feed resources and contribution to soil fertility in mixed crop-livestock production systems. To compare methods used by soil scientists and animal-nutritionists to assess quality of plant materials, three woody tropical legumes with contrasting qualities were used: Indigofera zollingeriana Miq. (Indigofera), Cratylia argentea Benth. (Cratylia) and Calliandra houstoniana (Mill.) Stan. var. calothyrsus (Meiss.) Barn. CIAT 20400 (Calliandra). Plant material of each legume was used either fresh, freeze-dried, frozen, oven-dried (60 °C) or air-dried in order to estimate extents and rates of aerobic degradation in litterbags on the soil during 140 days and anaerobic degradation in an in-vitro gas production experiment during 144 h. Results showed, that aerobic decomposition rates of leaf tissues were highest for Indigofera (k = 0.013 day⁻¹), followed by Cratylia (k = 0.004 day⁻¹) and Calliandra (k = 0.002 day⁻¹). Gas production rates evaluated under anaerobic conditions, were highest for Indigofera (k = 0.086 h⁻¹), intermediate for Cratylia (k = 0.062 h⁻¹) and lowest for Calliandra (k = 0.025 h⁻¹). Decomposition and gas production rates differed (P < 0.001) among species. Differences between post harvest treatments were not statistically significant (P > 0.05). The extent of decomposition was highest for Indigofera (82.5%, w/w), followed by Cratylia (44.6%) and Calliandra (26.4%). The extent of gas production was highest for Indigofera (218.8 ml), followed by Cratylia (170.1ml) and Calliandra (80.1 ml). Extent of decomposition and extent of gas production were significantly different (P < 0.001) among species. In contrast to the extent of decomposition, the extent of gas production was affected (P < 0.001) by sample post harvest treatments. Highest gas production was observed for the fresh and frozen treatments. The forage quality parameters that best correlated with aerobic and anaerobic degradation were lignin+bound condensed tannins, lignin+total condensed tannins/N, indigestible acid detergent fibre (IADF) and in-vitro dry matter digestibility (IVDMD). Results showed that differences in decomposition and digestibility were more related to intrinsic plant quality parameters than to changes in tissue quality induced by post harvest treatments. In addition, we found that rate of aerobic degradation of legume leaves on the soil was highly correlated (r > 0.80, P < 0.001) to IVDMD and gas production (r = 0.53, P < 0.001). These results indicate that plant measurements (IADF, IVDMD and gas production) used to assess forage quality in animal nutrition studies are more rapid and resource saving predictors for aerobic decomposition of tropical legumes than initial plant quality ratios (lignin+polyphenols/N and lignin+total condensed tannins/N) commonly used by many researchers. Furthermore, this study confirms the potential usefulness of IVDMD for screening tropical legumes for soil fertility management.     

The Effect of Mixing Prunings of Two Tropical Shrub Legumes (Calliandra Houstoniana and Indigofera zollingeriana) with Contrasting Quality on N Release in the Soil and Apparent N Degradation in the Rumen

Lack of synchronization between N released from prunings applied to the soil as green manures and crop uptake as well as optimization of protein digestibility for ruminants, remain major research objectives for the selection of multipurpose tree and shrub legumes (MPT) for mixed smallholder systems in the tropics. Prunings of the high tannin, low quality MPT Calliandra houstoniana CIAT 20400 (Calliandra) and the tannin free, high quality MPT Indigofera zollingeriana (Indigofera) were mixed in the proportions 100:0, 75:25, 50:50, 25:75, and 0:100 (w/w) in order to measure the aerobic rate and extent of N release in a leaching tube experiment, and the anaerobic extent of N degradation in an in vitro gas production experiment. Parameters measured in Calliandra:Indigofera mixtures were compared to theoretical values derived from single species plant material (i.e. 100:0 and 0:100). Aerobic N release and apparent anaerobic N degradation increased with increasing proportion of the high quality legume (Indigofera) in the mixture. While N release in the soil was lower than theoretical values in the mixture 50% Calliandra/50% Indigofera, this was not the case with apparent anaerobic N degradation with the same mixture. Aerobic N immobilization was more pronounced for the mixture 75% Calliandra/25% Indigofera than for 100% Calliandra and negative interaction was observed with apparent anaerobic N degradation in the mixture 75% Calliandra/25% Indigofera. Plant quality parameters that best correlated with aerobic N release and apparent anaerobic N degradation in the rumen were lignin + bound condensed tannins (r=−0.95 and −0.95 respectively, P<0.001). In addition, a positive correlation (r=0.89, P<0.001) was found between aerobic N release in the leaching tube experiment and apparent N degradation in the in vitro anaerobic gas production experiment. Results show that mixing prunings of MPT materials with contrasting quality is an effective way to modify aerobic N release pattern as well as apparent anaerobic N degradation and could possibly be applied to minimize N losses in the rumen and in the soil. In addition, apparent anaerobic N degradation was identified as good predictor of aerobic N release in the soil, which has resource saving implications when screening MTP to be used as green manures.     

Distribution and diversity of rhizobia nodulating agroforestry legumes in soils from three continents in the tropics

The natural rhizobial populations of Calliandra calothyrsus, Gliricidia sepium, Leucaena leucocephala and Sesbania sesban were assessed in soils from nine sites across tropical areas of three continents. The rhizobial population size varied from undetectable numbers to 1.8 x 104 cells/g of soil depending on the trap host and the soil. Calliandra calothyrsus was the most promiscuous legume, nodulating in eight soils, while S. sesban nodulated in only one of the soils. Polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analyses of the 16S rRNA gene and the internally transcribed spacer (ITS) region between the 16S and 23S rRNA genes were used to assess the diversity and relative abundance of rhizobia trapped from seven of the soils by C. calothyrsus, G. sepium and L. leucocephala. Representatives of the 16S rRNA RFLP groups were also subjected to sequence analysis of the first 950 base pairs of the 16S rRNA gene. Eighty ITS groups were obtained, with none of the ITS types being sampled in more than one soil. RFLP analysis of the 16S rRNA yielded 23 'species' groups distributed among the Rhizobium, Mesorhizobium, Sinorhizobium and Agrobacterium branches of the rhizobial phylogenetic tree. The phylogeny of the isolates was independent of the site or host of isolation, with different rhizobial groups associated with each host across the soils from widely separated geographical regions. Although rhizobial populations in soils sampled from the centre of diversity of the host legumes were the most genetically diverse, soil acidity was highly correlated with the diversity of ITS types. Our results support the hypothesis that the success of these tree legumes in soils throughout the tropics is the result of their relative promiscuity (permissiveness) allowing nodulation with diverse indigenous rhizobial types.     

N recovery from legume prunings and priming effects are governed by the residue quality

Nitrogen recovery from 15N-labelled prunings of Gliricidia sepium, Peltophorum dasyrrachis, Calliandra calothyrsus and Leucaena leucocephala, each of two different chemical qualities, was followed over three cropping cycles in a growth room. Half of the pots of each treatment received a further addition of unlabelled pruning material, from the same species as that previously applied, before the second and third crop cycle. The cumulative maize total N accumulation revealed the largest benefit from N rich, low lignin and polyphenols Gliricidia prunings followed by Leucaena, Calliandra and Peltophorum. Cumulative N recovery measured using 15N over the three crop cycles ranged from 9% from Calliandra prunings to 44% from Gliricidia prunings. The vast majority of this N was recovered during the first crop cycle which agreed well with estimates using the N difference method. Recoveries in the second and third crops ranged from 0.4–5% (15N method) and 6–14% (N difference method) of the N initially applied. The protein binding capacity of polyphenols was the best predictor of N recovery at both initial and later crop cycles. Treatments which led to a large N recovery initially, continued to provide greater N benefits in subsequent cycles although with increasing harvest time this trend decreased. Thus, there was no compensation in initial N release from low quality prunings at later harvests and the agronomic implications of this are discussed. Addition of unlabelled Gliricidia prunings before the second and third cycle led to a positive apparent priming effect on previously applied 15N labelled prunings. By contrast, repeated additions of Peltophorum residues, rich in lignin and active polyphenols, resulted in a reduced recovery of initially applied pruning-15N. However, the maximum positive or negative effects on recovery of pruning N amounted to less than 2% recovery of the initial amount of N added over 14 weeks. Thus the scope for regulation of N release from tree prunings during these later stages of decomposition appears to be limited.     

Application of 15N and xylem ureide methods for assessing N2 fixation of three shrub legumes periodically pruned for forage

The apparently diminished capacity for N₂ fixation by the shrub legume Calliandra calothyrsus (Calliandra) relative to other woody perennial legumes was investigated in a field experiment in northern Queensland, Australia. In this trial, (i) the proportion of plant nitrogen (N) derived from symbiotic N₂ fixation (%Pfix) and the amounts of N₂ fixed were compared in Calliandra, Gliricidia sepium (Gliricidia) and Codariocalyx gyroides (Codariocalyx), (ii) variations in N₂ fixation due to season or tree age were determined, (iii) estimates of Pfix derived with the ¹⁵N natural abundance technique were compared with values obtained from ¹⁵N enrichment or xylem sap ureide procedures to determine whether the previous conclusions about Calliandra's ability to fix N had resulted from specific problems with the natural abundance methodology used in the earlier studies.Inoculated seedlings of each of the three shrub legume species were planted in dense stands (1.5 m rows, 0.5 m between trees) in two randomised blocks. The northern block was used solely for natural abundance measurements, while ¹⁵N-enriched KNO₃ (10 atom % ¹⁵N excess) was applied four times over a 52 week period to plots in the southern block. The non-nodulating tree legume Senna spectabilis (formally Cassia spectabilis) was used as a non-N₂-fixing reference for the ¹⁵N-based procedures, with Guinea grass (Panicum maximum) included as an additional non-fixing check. Growth by the trees above 75 cm was first cut and removed after 22 weeks and regrowth was subsequently pruned periodically for another 95 weeks. Sampling for dry matter production, N yield and estimates of Pfix were restricted to the central four of the 32 plants which constituted each replicate plot. Information generated during the 117 week study indicated that estimates of Pfix by ¹⁵N natural abundance were closely similar to values derived with ¹⁵N-enrichment or sap ureides. The data indicated that Calliandra had a reduced reliance upon N₂ fixation relative to Gliricidia and Codariocalyx for the first 65 weeks after establishment. This appeared to be due to more prolifc root growth by Calliandra than either of the other N₂-fixing species and an ability to extract a greater proportion of its N requirements from soil mineral N. However, after week 65 and for the remainder of the experiment, estimates of Pfix for Calliandra were similar to the other shrub legumes. Over 117 weeks, prunings from Calliandra and Gliricidia had removed 52–58 t dry matter ha^-1, and between 1471 and 1678 kg N ha^-1, of which 1026–1063 kg N ha^-1 was estimated to have been derived from N₂ fixation. At the time of final harvest, 65–73% of the fixed N was present in shoot regrowth of the N₂ fixing shrubs, 9–18% in the roots, 15% in the trunk, and 2–6% in fallen leaves.     

Isozyme variation in Calliandra calothyrsus (Leguminosae): its implications for species delimitation and conservation

Patterns of genetic diversity within and among populations of Calliandra calothyrsus, an important multipurpose tree species, were examined using isozyme analysis. C. calothyrsus is a widespread species distributed throughout Central America and southern Mexico, across a variety of environments. Morphologically and ecologically distinct populations can be identified within this range, but they are currently considered to represent a single species. C. calothyrsus has been introduced to many parts of the tropics, where it is cultivated as a source of fuelwood, animal fodder, green manure, and shade by rural communities. Some of these introductions are known to have originated from Guatemala, but very little is known about the genetic diversity of either the native or naturalized populations. Isozyme electrophoresis of 23 loci across 17 populations of C. calothyrsus indicated that the majority of genetic diversity was partitioned between populations (FST = 0.802) and that within-population heterozygosity was low (mean Ho = 0.057). Naturalized populations had lower than expected heterozygosities and were most similar to material from Santa Maria de Jesus, a natural population in southern Guatemala. Four distinct groups of populations were identified on the basis of Nei's genetic distances and Population Aggregation Analysis (PAA), and correlate with the morphological and ecological differences that can be observed within the species. The results are discussed in relation to species delimitation and conservation.     

A new calymmate mimosoid polyad from the Miocene of Argentina

A palynomorph with an unequivocal relationship to the eight-grain polyads of the mimosoid genus Calliandra Benth., is described from the Miocene sediments of San Juan Province, Argentina. Comparison of the fossil palynomorph with polyads of the extant Calliandra species shows a resemblance to those which have one, highly specialized, appendiculate monad. The new palynomorph has a rudimentary appendix, apparently transitional in the path leading to the more highly developed appendiculate forms in the extant Calliandra group. This specialized polyad type is considered to be one of the most highly evolved forms in subfamily Mimosoideae. The closest affinity of the new fossil polyad is with the eight-grain calymmate polyads of Calliandra chilensis Benth., a species which has developed in the extra-tropical, xerophilous shrub, habitat of north-central Chile. The disappearance of Calliandra species in San Juan Province is thought to be related to the culmination of the Andean rising, and the consequent interruption to the Pacific Ocean climatic influence. This new discovery is the first fossil record of Calliandra for Argentina, as well as being the most southerly and the oldest. It reinforces the hypothesis of an early origin and diversification for the Leguminosae in Tropical America.     

Mycorrhizas in agroforestry: spread and sharing of arbuscular mycorrhizal fungi between trees and crops: complementary use of molecular and microscopic approaches

The spread of arbuscular mycorrhizal (AM) fungi from tree to crop roots was examined by molecular and microscopic methods in a glasshouse study. Growth of Calliandra calothyrsus Meissner trees inoculated with isolates of the AM fungi Glomus etunicatum Becker and Gerdemann and Gigaspora albida Schenck and Smith was monitored over an 18-month period. Three successive ‘intercrops’ of beans or maize were sown at 25, 50 and 75 cm distances from the tree and harvested during this period. At each crop harvest, the distribution of tree and crop roots and the spread of the inoculant fungi were determined using traditional microscopic methods and fungal specific primers. Both inoculants greatly improved the growth of the trees and colonization spread to the crops once the trees were 6 months old. However, benefits of inoculation to crop growth were not observed due to increased competition from the larger inoculated trees growing in a restricted soil volume. Of the two inoculant fungi, Glomus etunicatum appeared to be more mobile as it spread more rapidly, formed higher levels of colonization at increasing distances from the tree and was responsible for most of the mycorrhizal cross-contamination. In contrast, colonization of tree and crop roots by Gigaspora albida was higher nearest the tree. This work demonstrated the benefits of mycorrhizal fungus inoculation for tree growth and confirmed that trees and crops share the same AM fungi. Trees may therefore act as reservoirs of mycorrhizal fungi, either inoculant or indigenous, for surrounding crops or other annual vegetation. It was also shown that tree pruning, the normal practice in agroforestry systems, did not reduce mycorrhizal colonization or prevent spread to crops. However, the slow rates of inoculant spread found here suggest that it may take years before inoculants benefit the growth of crops sown several metres from the tree. The work also demonstrated that microscopic quantification of mycorrhizal colonization and the use of molecular probes to identify specific fungi within roots can complement each other effectively. Molecular probes were more sensitive at detecting mycorrhizal fungi than microscopic methods, but did not discriminate between full mycorrhizal structures and traces of hyphae.     

Calliandra dolichopoda and C. cualensis (Leguminosae, Mimosoideae), two new species from Mexico

Calliandra dolichopoda and C. cualensis, two species endemic to Guerrero and Jalisco, Mexico, respectively, are described and illustrated. Both species appear to be closely related to C. hirsuta.

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Resumen  Se describen e ilustran Calliandra dolichopoda y C. cualensis, dos especies endémicas de Guerrero y Jalisco, México, respectivamente. Ambas especies parecen estar relacionadas con C. hirsuta.

     

Fungitoxic effects of nonprotein imino acids on growth of saprophytic fungi isolated from the leaf surface of Calliandra haematocephala

Four saprophytic and pathogenic fungi were isolated from the leaf surface of Calliandra haematocephala, a tropical legume known to contain large amounts of rare nonprotein imino acids in its leaves and seeds. The fungi Aspergillus niger, Aspergillus sp., Curvularia sp., and Penicillium sp. were cultured in the laboratory and tested for susceptibility to leaf extracts of the host plant and to proline, pipecolic acid, cis-5-hydroxypipecolic acid, and 2,4-trans-4,5-cis-4,5-dihydroxypipecolic acid. Fungal spore germination and germ tube growth were measured. Aspergillus sp. was inhibited by plant extracts and by pipecolic acid and cis-5-hydroxypipecolic acid. Curvularia sp. growth was stimulated by plant extracts and by pipecolic acid. The other two fungi were unaffected by any of the treatments. The data indicate that imino acids may play a role in the specific resistance of Calliandra spp. to Aspergillus sp.     

Calliandra anthoniae (Leguminosae,Mimosoideae, Ingeae), a new species,and a new combination in Pseudopiptadenia Rauschert (Leguminosae,Mimosoideae, Mimoseae)

Calliandra anthoniae is described and illustrated, and the combinationPseudopiptadenia suaveolens is made.     

Fungitoxic effects of nonprotein imino acids on growth of saprophytic fungi isolated from the leaf surface of Calliandra haematocephala.

Four saprophytic and pathogenic fungi were isolated from the leaf surface of Calliandra haematocephala, a tropical legume known to contain large amounts of rare nonprotein imino acids in its leaves and seeds. The fungi Aspergillus niger, Aspergillus sp., Curvularia sp., and Penicillium sp. were cultured in the laboratory and tested for susceptibility to leaf extracts of the host plant and to proline, pipecolic acid, cis-5-hydroxypipecolic acid, and 2,4-trans-4,5-cis-4,5-dihydroxypipecolic acid. Fungal spore germination and germ tube growth were measured. Aspergillus sp. was inhibited by plant extracts and by pipecolic acid and cis-5-hydroxypipecolic acid. Curvularia sp. growth was stimulated by plant extracts and by pipecolic acid. The other two fungi were unaffected by any of the treatments. The data indicate that imino acids may play a role in the specific resistance of Calliandra spp. to Aspergillus sp.     

Nitrogen and Phosphorus Release from Decomposing Leaves under Sub‐Humid Tropical Conditions1

For many soils of the tropics, inputs of organic materials are essential to sustain soil fertility and crop production. Research in the quality of organic inputs, a key factor controlling rates of decomposition and nutrient release, continues to guide selection and use of organic materials as nutrient sources. The relationship between decomposition patterns and the quality parameters of the fresh leaves of six agroforestry species: Sesbania sesban, Croton megalocarpus, Calliandra calothyrsus, Tithonia diversifolia, Lantana camara, and Senna spectabilis, was investigated in a litterbag study over a period of 77 days in the highlands of western Kenya. The litterbags were buried 1 cm below the soil surface and covered with soil of ca 1 cm thickness. Percent leaf mass and total N and P that remained with time strongly correlated with total P and C/P ratio (R²= 0.60‐0.90) during the first 35 days of study; but afterwards, correlation was stronger with the initial soluble polyphenolics (Pp)/P ratio (R²= 0.69‐0.92) than with total P and C/P ratio. Loss of leaf mass and release of N and P followed the exponential function, y_t= y₀* e‐^kt, from which the specific decay rate constants (k) were calculated for loss of leaf mass (k_B) and release of N (k_N) and P (K_p). Among the plant species, the k values were lowest in Calliandra with k_B= 0.012/d, k_N= 0.017/d and k_p= 0.044/d. Lantana had the highest K values with k_g= 0.067/d and k_p= 0.119/d, but the highest k_N value of 0.109/d occurred in Tithonia. The k_B values for all organic materials were lower than their corresponding k_N and k_p values, suggesting that leaching of N and P from litters may have augmented the microbial mineralization of N and P. There was a strong correlation between the k_B, k_N, and k_p values and total P (r = 0.82‐0.96; P 0.01), but not total N, lignin (LIG), or Pp. Rates of N and P release followed the general trend: Tithonia > Senna > Lantana > Sesbania > Croton > Calliandra. The results indicated that, among the quality parameters studied, total P is the most important factor controlling rate of decomposition and N and P release from organic inputs in the area of study.     

2S,4R-carboxy-2-acetylamino-4-piperidine dans les feuilles de Calliandra haematocephala

A new naturally occurring pipecolic acid derivative has been isolated from leaves of the legume Calliandra haematocephala. Its structure was shown to be 2S,4R-carboxy-2-acetylamino-4-piperidine by chemical and spectrosopic methods.     

Effect of the tropical forage calliandra on microbial protein synthesis and ecology in the rumen

AIMS: To determine the effect of condensed tannins in Calliandra calothyrsus (calliandra) on rumen microbial function. METHODS AND RESULTS: Microbial populations, ruminal protein synthesis and fermentation end-products were measured in sheep fed roughage hay supplemented with calliandra (30%), with and without inclusions of polyethylene glycol (PEG) to counteract the effect of tannin. Molecular and conventional enumeration techniques were used to quantify rumen bacteria, fungi and protozoa, and protein synthesis was predicted from estimates of urinary purine excretion. The total number of cellulolytic bacteria, including populations of Fibrobacter succinogenes and Ruminococcus spp., was significantly lower in sheep supplemented with calliandra and these populations increased when animals were treated with PEG. By contrast, protozoa and fungi and the microbial group containing Bacteroides-Porphyromonas-Prevotella bacteria appeared to be less affected. The efficiency of microbial protein synthesis in the rumen was not altered significantly. CONCLUSION: Calliandra caused significant shifts in rumen microbial populations without changing the efficiency of protein synthesis. SIGNIFICANCE AND IMPACT OF THE STUDY: The effect of calliandra tannins on rumen digestion may result more from complexing with nutrients than direct inhibition of micro-organisms.     

Cis-4-hydroxypipecolic acid and 2,4-cis-4,5-trans-4,5-dihydroxypipecolic acid from Calliandra

cis-4-Hydroxypipecolic acid and 2,4-cis-4,5-trans-4,5-dihydroxypipecolic acid were isolated from leaves of Calliandra pittieri. A system for resolving the eight imino acids isolated from Calliandra is described.     

2,4-cis-4,5-cis-4,5-Dihyroxypipecolic acid—a naturally occurring imino acid from Calliandra pittieri

2,4-cis-4,5-cis-4,5-Dihydroxypipecolic acid has been isolated from the leaves of Calliandra pittieri. This is the third dihydroxypipecolic acid isomer isolated from Calliandra and the first report of this compound from a natural source.     

Effect of the tropical tannin-rich shrub legumes Calliandra calothyrsus and Flemingia macrophylla on methane emission and nitrogen and energy balance in growing lambs

The objective of this study was to test whether the use of tannin-rich shrub legume forage is advantageous for methane mitigation and metabolic protein supply at unchanged energy supply when supplemented in combination with tannin-free legumes to sheep. In a 6 × 6 Latin-square design, foliage of two tannin-rich shrub legume species (Calliandra calothyrsus and Flemingia macrophylla) were used to replace either 1/3 or 2/3, respectively, of a herbaceous high-quality legume (Vigna unguiculata) in a diet composed of the tropical grass Brachiaria brizantha and Vigna in a ratio of 0.55 : 0.45. A Brachiaria-only diet served as the negative control. Each experimental period lasted for 28 days, with week 3 serving for balance measurement and data collection inclusive of a 2-day stay of the sheep in open-circuit respiration chambers for measurement of gaseous exchange. While Vigna supplementation improved protein and energy utilisation, the response to the partial replacement with tannin-rich legumes was less clear. The apparent total tract digestibilities of organic matter, NDF and ADF were reduced when the tannin-rich plants partially replaced Vigna, and the dose-response relationships were mainly linear. The tannin-rich plants caused the expected redistribution of more faecal N in relation to urinary N. While Flemingia addition still led to a net body N retention, even when fed at the higher proportion, adding higher amounts of Calliandra resulted in body protein mobilisation in the growing lambs. With respect to energy, supplementation of Vigna alone improved utilisation, while this effect was absent when a tannin-rich plant was added. The inclusion of the tannin-rich plants reduced methane emission per day and per unit of feed and energy intake by up to 24% relative to the Vigna-only-supplemented diet, but this seems to have been mostly the result of a reduced organic matter and fibre digestion. In conclusion, Calliandra seems less apt as protein supplement for ruminants while Flemingia could partially replace a high-quality legume in tropical livestock systems. However, methane mitigation would be small due to associated reductions in N and energy retention.     

2,4-Trans-4,5-trans-4,5-dihydroxypipecolic acid and cis-5-hydroxypipecolic acid from leaves of Calliandra angustifolia and sap of C. Confusa

2,4-Trans-4,5-trans-4,5-dihydroxypipecolic acid and cis-5-hydroxypipecolic acid have been isolated from the leaves of Calliandra angustifolia and the sap of C. confusa. Distribution of these and other non-protein amino acids is discussed.