Differences in Root Systems Among Common Genera of Orchidaceae in a Tropical Cloud Forest Taryn Sena Dunivant Plant Science University of California, Santa Cruz EAP Tropical Biology and Conservation, Fall 2016 16 December 2016 Abstract Most tropical forest s consist of a den se stem count, presenting many opportunities for epiphytes to find a niche and diversify Orchidaceae is a strong example of biodiversity in a tropical cloud forest which is supported by representing one of the largest families within angiosperms and containing countless epiphytic species. Their unique abilities to survive as epiphytes are showcased in their complex root systems. A key structure to these impressive root systems are the outermost layer termed the velamen. The velamen is comprised of spongy dead cells ranging from one to eighteen cells thick. This sheath like layer serves as a source of protection and absorption for the interior of the system, and consequently the overall fitness of the plant Examining these root systems among genera and relating them to each other may give insight to how closely similar or different the root systems are between ge nera Six common orch id genera ( Lepanthes Restrepia Masdevallia Epidendrum Maxillaria Oncidium ) of Monteverde, Costa Rica were selected and their root systems were st udied. I took measurements of the root diameter, velamen thickness, and the number of cells thick the velamen was. Overall there was a positive correlation found between the root diameter and the thickness of the velamen, though not in all genera (e.g Epidendrum ). Correspondingly, as the t hickness of the velamen increases, the number of cells increases. Orchidaceae root systems have very little variation in some genera and in others are widely variable This study shows how the root systems of Orchiaceae can differ both between and within genera. Diferencias del sistema radical de entre g ÂŽ neros de orquÂ’ deas communes d el bosque tropic a l nuboso Resumen La mayor parte de los bosques tropicales presentan una alta abundancia de Â‡rboles, presentando asÂ’ muchas oportunidades para que plantas epÂ’fitas encuentren un nicho y diversifiquen. Orchidaceae es un ejemplo de biodiversidad en el bosque tropical nuboso, representando la familia de angiospermas mÂ‡s diversa. Los complejos sistemas radicales de las orquÂ’deas juegan un papel muy importante en la adaptaciÂ—n de estas para una vida epÂ’fita. Una estructura clave dentro de estos sistemas es la capa exterior de las raÂ’ces, conocida como velamen. El velamen estÂ‡ compuesto por cÂŽlulas esponjosas muertas, y puede presentar de una a
Differences in Orchidaceae Root Systems Dunivant 2 18 cÂŽlulas de grosor. Esta capa de cÂŽlulas contribuye a la protecciÂ—n de la raÂ’z, asÂ’ como a la absorciÂ—n de nutrientes. El objetivo princi pal de este estudio es investigar y comparar el vel amen de seis diferentes gÂŽneros comunes de orquÂ’deas ( Lepanthes Restrepia Masdevallia Epidendrum Maxillaria Oncidium ) que se encuentran en el bosque tropical nuboso. MedÂ’ el diÂ‡metro de las raÂ’ces, grosor del velamen y el nÂœmero de cÂŽlulas de grosor del velamen. En general encontrÂŽ una correlaciÂ—n positiva entre el diÂ‡metro de las raÂ’ces y el grosor del velamen, sin emb argo no todos los gÂŽneros presentaron esta correlaciÂ—n (por ejemplo Epidendrum ). AdemÂ‡s el grosor del velamen incrementa con el nÂœmero de cÂŽlulas de grosor del mismo. Algunos gÂŽneros de orquÂ’deas presentaron muy poca variaciÂ—n en sus sistemas radicales mie ntras que otros presentaron mucho mÂ‡s. Este estudio muestra cÂ—mo los sistemas de raÂ’cea de Orchidaceae pueden diferir entre y dentro de gÂŽneros. Introduction Orchid aceae is one of the largest families with in angiosperms and contains nearly 27, 800 known species worldwide. The Monteverde region of Costa Rica contains over 500 Orchidaceae species, which is approximately 2% of the world 's orchid species and 39% of Costa Rica's orchid species This is the highest diversity found within Orchidaceae in any co mparable neotropical region and is due to the many life zones in Monteverde, providing a variety of habitats (Atwood, 2000) Orchids are exceptionally diverse within the family and range in living conditions from tree canopies in tropical zones to grassla nds of temperate zones. Epiphytic species living in tree canopies have had opportunities to find new niches and diversify. The evolutionary pathway for this diversification could have occurred differently between Orchidaceae genera (Nepokroeff and Sytsma, 2000). D iversity among orchids is found in the uniqueness of their structures from the inflorescences reproductive structures, to the cells of the root systems Orchids are monocots and therefore never contain a taproot, however are comprised of secon dary roots that extrude from the stem (Dressler 1981). The root systems of orchids are important for the epiph ytic species because they are the adhesi on basis to the host plant and the attachment source to tree trunks and branches need t o be flexible and strong. The root systems are vulnerable to environmental conditions as well as many other o rganisms living in the canopy. Root systems of vascular plants are their life source, with the main two functions being anchorage and absorption, providing wate r, nutrients, stability and are more extensive then the vegetative growth. Orchidaceae root systems are also a photosynthetic organ, there are a number of orchid species that do not grow any vegetation and rely on the roots alone to produce the sugar sour ce. Also the vegetation can be small and considering the varying light levels of living in the canopy, it would be efficient for m ultiple organs to photosynthesize Most importantly, root systems are the primary source for water and nutrients. The root s ystem is made up of a variety of plant tissues. The center of the root is known as the vascular cylinder, which contains the vascular tissues, phloem and xylem, as well as a non vascular tissue, the pericycle A compact layer of cells surrounding the vas cular cylinder is known as the endodermis, serving as a line o f defense Following is the cortex comprised of cortica l tissue and is responsible for the major of the root body. The cells of the cortex are loosely a rranged providing spaces essential aera tion (Raven, 1999) They are also the cells that house the chloropl asts for photosynthetic roots. Defending the cortex is the last layer, the epidermis In the family Orchidaceae, most orchids
Differences in Orchidaceae Root Systems Dunivant 3 have an additional layer named the velamen (Figure 1.V) This layer is not specific to just orchids but is found primarily throughout the family and other plants that do possess velamen are closely related (Dressler, 1981) The velamen is a specialized tissue, composed of cells that are dead at maturity and can therefore absorb large amounts of water, working like a sponge. This modified spongy epidermis can be one to eighteen cells thic k (Dressler 1981) The outermost layer of the velamen cells can develop root hairs (Dycus and Knudson, 1957). T he true function of the velamen has been debated and many scientists have developed several conclu sions. Many are certain the velamen is solely for water catchment because it is important for aerial plants that are not living in a substrate to have multiple ways to absorb and hold onto water and nutrients. Not only could the velamen help to collect water, but also to reduce the loss of water. The velamen as well serves as a mechanical protection source, working as a sheath, to guard the photosynthetic root system from the environmental conditions (RÂŽpanier et al., 2009). Others believe that it is r elated to the mycorrhizal relationship between orchi ds and fungi, since many fungi reside in the empty velamen cells. The mycorrhizae in orchids are important as a nutrient source and even for germination success. With Orchidaceae being a fantastic example of biodiversity, niche partitioning and complex anatomy, it is significant to investigate the variability in their root systems. I want to explore the root systems of orchids in Monteverde, Costa Rica, where the diversity of or chids is plentiful and unlike no other in the neotropics I have selected six common genera within Orchidaceae ( Lepanthes, Restrepia, Masdevallia, Epidendrum, Maxillaria, and Oncidium) all with in the sub family Epidendreae. I will explore the question: W hat are the differences in the root systems of orchids among genera in a tropical cloud forest? Figure 1 Freehand cross section of an Orchidaceae root at 40x. ( V ) Velame n layer surrounding the root system. V
Differences in Orchidaceae Root Systems Dunivant 4 Materials and Methods I collected Orchi daceae specimens from the Estaci Â—n BiolÂ— gica Monteverde Reserve. Samples were obtained from fallen tree trunks, branches, and moss piles. I identified the specimen to genu s level and tagged them Six genera were selected b ased on the abundance found, which include: Lepanthes, Restrepia, Masdevalli a, Epidendrum, Maxi llaria, and Oncidium. I could not identify s p ecimens to species level due to lack of flowers therefore the richness of each genus is not known Fo r each specimen, three healthy roots were sampled randomly and a freehand cross section was prepared as adjacent to the base of the root as possible which is the oldest segment of the root A compound microscope ( Olympus CX22LED ) equipped with a n ocular micrometer was used to measure the root diameter, the thickness of the velamen, and the number of cells thick contained within the vela men I noted a ny interesting characteristics such as coloration, root hairs, or cell configuration Results Sample sizes varied between 13 22 specimen, per genus, therefore samples measured varied between 39 66 cross sections per genus. I calculated the rario of the velamen thickness to the root diameter Epidendrum showed the largest root diameter while Maxillaria showed the largest velamen thickness. The highest ratio between velamen thickness and root diameter was found in Oncidium and the lowest in Lepanthes (Table 1). Table 1. Average ( SD) of root diameter, velamen thickness, number of cells in velamen thickness and velamen root ratio, in orchid specimens collected in Monteverde, Costa Rica Genus n Root Diameter (m) Velamen Thickness (m) Number of Cells in Velamen Velamen/ Root Diameter Lepanthes 20 650 165 41 10 1 0.064 0.018 Restrepia 22 565 98 53 12 2 0.095 0.022 Masdevallia 13 735 215 121 32 3 1 0.169 0.039 Epidendrum 18 1555 739 99 57 2 1 0.069 0.033 Maxillaria 19 840 472 162 128 3 1 0.187 0.068 Oncidium 21 854 345 159 80 4 1 0.189 0.071 The cross sections of genera revealed individuality among their root systems. The genus Lepanthes contained purple pigment in each sample observed as well as a velamen layer comprised of a single cell (Fig. 2A ). The velamen thickness of Lepanthes was the thinnest of the six genera. Restrepia contained large visible chloroplasts and the velamen was n ever more than two cells thick, and only occasionally contained a single cell in the velamen layer (Fig. 2B ). Restrepia had the smallest root diameter of the six. Masdevallia contained visible chloroplasts and usually had a distinctive three celled velam en layer (Fig 2C ). Epidendrum had clearly distinct cells and occasionally a ring or area of purple pigment (Fig. 2D ). The root diameter of Epidendrum was the largest, however had a low velamen thickness in comparison. Maxillaria had the thickest velame n layer and varied widely, the velamen layer was anywhere from three to six cells thick Maxillaria ranged in color from white, gold, to brown (Fig. 2E ). Oncidium also varied widely and the velamen contained anywhere from one to six cells. Oncidium had the
Differences in Orchidaceae Root Systems Dunivant 5 greatest percentage velamen thickness to the root diameter. This genus had pronounced thick root hair s on many of the samples (Fig. 2F ). Figure 2 Freehand cross sections of Orchidaceae root systems taken closest to the base as possible. Images taken from different magnifications 40x 100x from a compound microscope. ( A ) Lepanthes cross section shown at 100x. Distinct purple pigment and the velamen is comprised of a single cell layer ( B ) Restrepia cross section shown at 100x. Distinct green chloroplasts and velamen layer comprised of two cells thick. ( C ) Masdevallia cross section shown at 100x. Distinct green chloroplasts and a velamen layer comprised of three cells thick. ( D ) Epidendrum cross section shown at 40x. Distinct purple ring, green chloroplasts, and a velamen layer comprised of five cells thick. ( E ) Ma xillaria cross section shown at 40x. Distinct gold coloration and a velamen layer comprised of three cells thick. ( F ) Oncidium cross section shown at 40x. Distinct root hairs and a velamen layer comprised of four cells thick. 100x 100x 100x 4 0x 4 0x 4 0x A B C D E F
Differences in Orchidaceae Root Systems Dunivant 6 Figure 3 Velamen th ickness in contrast to the root diameter of six orchid genera. There are similarities in Lepanthes and Restrepia regarding the relationship between the thickness of the velamen layer and the diameter of the root both of these genera are mo re constant from species to species represented by the clusters Oncid ium Max illaria and Epidendrum are mor e diverse from species to species, represented by the spread values Also, Epidendrum has a consistent velamen thickness regardless of the diameter of the root diameter. Oncidium Maxillaria and Masdevallia display ed a pattern, as the root diameter increased the velamen thickness also increased (Fig. 3) "! #""! $""! %""! &""! '""! "! '""! #"""! #'""! $"""! $'""! %"""! %'""! ()*+,)-!./012-)33!4 5 ,6! 7889!:0+,)9);!4 5 ,6! !"#$%$&'( )*+$,,*-$*( ./$%0"%-&'( )*1%02*,,$*( 3014-0/$*( 50/*"4601(
Differences in Orchidaceae Root Systems Dunivant 7 Figure 4 N umber of cells thick with in the velamen layer compared to the thickness of the velamen. Regardless of the velamen thickness, Lepanthes was always one cell thick. Restrepia is one to two cells thick regardless of the thickness. However, the other four genera Oncidium, Maxillaria, Epidendrum, and Masdevallia showed a positive correlation between the velamen thickness and the number of cells. As the thickness of the velamen increased th e number of cells also increased (Fig. 4) Discussion Overall there is a positive correlation between the root diameter and the thickness of the velamen layer As the root diameter increases, the velamen thickness also increases. The same correlation can be observed with respect to velamen thickness and the number of cells thick the velamen is. As the v elamen thickness increases, the number of cells thick it is also increases. The similarity of trends between genera, seem s to represent the relatedness between the genera. W ithin the genera of Restrepia and Lepanthes there was little variation between all measurements Both of these genera are also similar in size structure. Restrepia had the lowest average of root diameter ( 565 98 m) and a low average percentage of velamen thickness to root diameter (9.5 2.2 %). Lepanthes had the lowest average percentage of velamen thickness to root diameter (6.4 1.8 %) and a small root diameter average ( 650 165 m) The two genera are small in overall size, with smaller vegetation and root structures This correlates to the taxonomic level of the two genera, they are both in the subtribe Pleurothallidinae. Also in this subtribe is Masdevallia which is the next closest relate d genus to these two based on the "! #! $! %! &! '! ?,@);!8A!B)**3! ()*+,)-!./012-)33!4 5 ,6! !"#$%$&'( )*+$,,*-$*( ./$%0"%-&'( )*1%02*,,$*( 3014-0/$*( 50/*"4601(
Differences in Orchidaceae Root Systems Dunivant 8 morphological results however it show cased more variab ility in root structural size. Unlike the low percentage found in Restrepia and Lepanthes Masdevallia had a high average percentage velamen thickness to root diameter (16.9 3.9 %). The genus Epidendrum is in the subtribe Laeliinae and seemed to follow a pattern of its own. Reg ardless of the root diameter, which varied widely ( 392 2210 m ) the velamen stay ed consistent within a small range ( 40 250 m ) This was made clear in the low average percentage of velamen to root diameter (6.9 3.3 %). Oncidium and Maxillaria are similar in the fact that they are both widely variable in root size structure B oth genera have well pronounced pseudobulbs and vary widely in vegetative morphological characteristics. Oncidium in the subtribe Oncidiinae and Maxillaria in the sub tribe Maxillariinae are the only two genera studied not in the tribe Epidendreae Both these genera are the only two that can be found outside the Neotropics and in the West Indies. Oncidium had the highest average percentage of velamen thickness to root diameter (18.9 3.9 %) as well as the largest average amount of cells that make up the velamen thickness (4 1) Maxillaria had the highest average of velamen thickness ( 162 128 m) as well as a high average percentage of velamen thickness to r oot diameter (18.7 6.8 %). Conclusions lead to an overall trend, as the root dimeter increase s the thickness of the velamen increases and as the velamen thickness increase s, the number of cells t hick it ha s also increases. G enera that have more overall variation also have more variation in their root system s Genera that have less variation overall within the genus are less variable in their root systems. Genera that are more c losely related taxonomically, have root systems that are more sim ilar. This study shows how the root systems of Orchiaceae can differ both between and within genera. Consideration needs to be taken in regards to the results. The age of each specimen or the exact level that they were originally inhabiting in the tree canopy was not known since specimens were collected off the ground. This could play a role in the variation within each genera. Also since the species could not be identified to species level and morph species could not be determined, the n umber of species within each genus is not known. Therefore, there could be a bias within a genus. The lack of variation in a genus may be due a lac k of species diversity collected. G enera with a higher variation may have had a high diversity of species within the sample. Identification to species level and the ability to collect these specimens from their physical locations will improve the study. Acknowledgments I would like to thank my primary advisor, Andres Camacho, for all his insight and hel p. My secondary advisor, SofÂ’a Acre Flores for guidance. The tiny lab squad, for keeping those long lab days rolling. Most importantly, Eladio Cruz for aiding me in orchid identification, answering all my questions, and allowing me to house my large orc hid collection on his property. Also thanks to the Cruz Obando family for housing me in their beautiful home.
Differences in Orchidaceae Root Systems Dunivant 9 Literature Cited Atwood, J. 2000. Orchids. Monteverde: Ecology and Conservation of a Tropical Cloud Forest Oxford University Press. New York. pp 74 75. Dressler, R. L. 1981. The Orchids: Natural History and Classification Harvard University Press. Cambridge. pp 1 2,25 31. Dycus, A., Knudson, L. 1957. The Role of the Velamen of the Aerial Roots of Orchids. Botanical Gazette. Chicago. Vol. 119.2: 78 87 Nepokroeff, M ., Sytsma, K. 2000. Evolution in Cloud Forest Psychotria Section Notopleura Via Growth Form Diversification. Monteverde: Ecology and Conservation of a Tropical Cloud Forest Oxford University Press. New York. pp 75 78. Raven, P., Evert R., Eichhorn, S. 1999. Biology of Plants W.H. Freeman and Company/worth Publishers. USA. pp 589 606. TrÂŽpanier M., Lamy M., Dansereau B. 2009. Phalaenopsis can absorb ure a directly through their roots. Plant Soil. Vol. 319: 95 100.