bs_bs_banner Botanical Journal of the Linnean Society, 2014, 176, 260–276. With 6 figures Structure and evolution of the pod in Indigofera (Fabaceae) reveals a trend towards small thin indehiscent pods VIBHA CHAUHAN and ARUN K. PANDEY* Department of Botany, University of Delhi, Delhi-110007, India Received 9 July 2013; revised 8 April 2014; accepted for publication 27 July 2014 Pod morphology and anatomy have been studied in 28 species and four varieties of Indian Indigofera. Pods of Indigofera spp. differ with respect to size, the relative thickness of the fruit wall, the number of sclerenchymatous layers in the endocarp, the presence/absence of hypodermis and trichomes, and the presence of separation tissue. Anatomically, the pericarp is broadly characterized into three types: type I (thin pericarp and three to five sclerenchymatous layers in the endocarp), type II (intermediary pericarp thickness and six to eight sclerenchymatous layers), and type III (thick pericarp and more than eight sclerenchymatous layers). The distribution of these types across the tribe is not congruent with the current phylogenetic analyses. Type III pericarp (present in the early diverging lineages of the tribe) represents the most primitive state, whereas type I and type II pericarps are derived. Fruits of Indigofera generally show normal explosive dehiscence as a means of dispersal of seeds, although some species show adaptations for dispersal by wind. In Indigofera, dehiscence is caused by a separation layer present at the dorsal and ventral sutures except in some species (Indigofera hochstetteri, Indigofera karnatakana, Indigofera glandulosa var. sykesii, and Indigofera trita var. scabra) in which no separation tissue is present; these species show delayed dehiscence or an indehiscent condition. The indehiscent pod type is considered to be apomorphic. The taxonomic, functional, and evolutionary significance of morphological and anatomical features in fruits of the genus Indigofera has been evaluated. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 176, 260–276. ADDITIONAL KEYWORDS: anatomy – dehiscence – Leguminosae – morphology. INTRODUCTION Indigofera L. (tribe Indigofereae, Fabaceae) includes 700–750 species (Schrire, 2005; Schrire et al., 2009). The genus is pantropical in distribution but the major centres of diversity are in Africa and Madagascar (550 species), the Sino-Himalayan region (105 species), and Australia (50 species); the remaining 45 species occur in the New World (Schrire et al., 2009). In India, the genus is represented by 60 species and 11 varieties, of which 16 species and seven varieties are endemic (Schrire, 1992; Sanjappa, 1995; Chauhan, Pandey & Schaefer, 2013). Indigofera spp. are annuals or perennials and habit varies from prostrate *Corresponding author. E-mail: arunkpandey@botany.du.ac.in; arunkpandey@hotmail.com 260 and erect herbs to under-shrubs, robust shrubs, and rarely trees (Fig. 1). Fruit morphology, anatomy, and dehiscence mechanisms in Fabaceae have been investigated in various studies (Fahn & Zohary, 1955; Esau, 1962; Fahn, 1967, 1982; Pate & Kuo, 1981; Kirkbride, Gunn & Weitzman, 2003). In Fabaceae, the pericarp consists of an exocarp (single or biseriate epidermal cell layers), mesocarp (multiple parenchyma and collenchymas cell layers), and endocarp (variable number of sclerenchyma cell layers). The mid region of the mesocarp is traversed by vascular strands. The endocarp is septate between seeds and often shows tannin spots. Usually, there is a relationship between the histological structure of the pericarp and the manner of fruit and seed dispersal (Guttenberg, 1971; Fahn & Werker, 1972). When the fruit matures, the two © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 176, 260–276 POD STRUCTURE AND EVOLUTION IN INDIGOFERA 261 Figure 1. Indigofera species showing habit, leaf types, flower and fruit. A, Indigofera oblongifolia. B, Indigofera trifoliata. C, Indigofera astragalina. D, Indigofera cassioides. E, Indigofera aspalathoides. F, Indigofera hendecaphylla. G, Indigofera dalzellii. valves of the dried legume usually twist. Dehiscence is brought about by the uneven shrinkage of the thickened walls of the pericarp cells. The greatest shrinkage occurs at right angles to the longitudinal axis of the fruit and microfibrils. The cell wall of the mature fruit dries and shrinks causing explosive dehiscence (Fahn & Zohary, 1955; Fahn, 1982). The mechanism of dehiscence and the function of separation tissue in the endocarp in the process of dehiscence were described by Fahn & Zohary (1955). Buckovic (1952) studied the anatomy of the legume pod and proposed that pod dehiscence was caused by different rates of moisture loss in different tissues, with the rate of loss being much higher in the paren- © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 176, 260–276 262 V. CHAUHAN and A. K. PANDEY chyma cells than in the fibrous cells. Fahn & Zohary (1955) studied the anatomy of the pod, particularly the pericarp structure, of > 100 species of Fabaceae. They recognized 17 types of legume based mainly on the structure of the sclerenchymatous layer of the pericarp. For dehiscence to occur, first the sclerenchymatous cells and/or the cellulose micelles in their cell walls must change orientation and, second, there must be separation tissue present extending into the region of the suture from the inner to the outer epidermis. The change in orientation leads to unequal swelling and shrinking of the cells in different directions, which in turn causes the pod to shatter. According to Roth (1977), this arrangement of fibres is responsible for the helicoidal bending of the valves. In other words, the axis of the bending movement (perpendicular to the long axis of the fibres) is oriented obliquely to the long axis of the fruits and this leads to helicoidal bending. Le Roux et al. (2011) concluded that a pattern of dehiscence in which, the orientation of the fibres in the endocarp relative to the longitudinal axis of the fruits is the major determinant for dehiscence to occur. Also, the presence of different zones of fibres and their orientation relative to one another are also important. Yang et al. (1990) observed and concluded that pod dehiscence was associated with the degree of mesocarp lignification. Gershon (1961) found that indehiscent species also exhibit decreased lignification of mesocarp cells, and observed that three indehiscent legume species all lacked separation tissues. Fahn & Zohary (1955) described the condition of not having a fibrous region at the sutures, and only thick-walled parenchyma without a separation layer, as most derived and thus considered indehiscence as a derived character in legumes. The species that lacked separation tissue in the pod are considered to be tardily dehiscent or indehiscent and are more derived compared to dehiscent pods. Because the phylogenetic relationships in Indigofereae are well known (Schrire et al., 2009), an opportunity exists to evaluate other useful taxonomic characters to infer the patterns of evolution. The present study aimed to: (1) to determine the taxonomic value of the morphological and anatomical structures of the pod in Indian Indigofera; (2) to trace the evolution of fruit characters in the tribe Indigofereae; and (3) to discuss the role of anatomical features in pod dehiscence and the mechanism involved. India. Voucher specimen information is provided with the author citation for these taxa in Table 1. Voucher specimens have been deposited in Delhi University Herbarium. POD MORPHOLOGY AND ANATOMY The length of mature pods (average of ten pods) was measured with the help of a scale. For anatomical studies, pods were fixed in formalin-acetic acidalcohol and dehydrated through an ethanol series (sensu Feder & O’Brien, 1968). Pods were cut into small pieces and were then processed in glycol methacrylate resin ‘Basic Resin’ infiltration. Sections, 2–3 μm thick, were cut on Microm HM 315 Thermo Scientific microtome and stained with aqueous toluidine blue and mounted in DPX. Photographs were taken with a microscope Primo Star Zeiss with a Canon digital camera (G10 14.7 MP). POD ANATOMICAL CHARACTERS AND CHARACTER STATES The sampled species belong to four clades: the Palaeotropical, Pantropical, Cape, and Tethyan clades of Indigofera (sensu Schrire et al., 2003, 2009) (Table 1). For character reconstruction, anatomical data were taken from our own observations and from the work of Leite et al. (2009) and Dave & Bennet (1990). A total of 41 accessions were included in the present study. Character states recorded for the pod anatomy include: pod type (I, II, III; coded as states 0, 1, 2, respectively) (Table 2). Phylogenetic trees were constructed using internal transcribed sequence data of the representative species using MrBayes (V. Chauhan & A. K. Pandey, unpubl. data). One hundred most probable trees were retrieved from the TRPROBS file produced in the Bayesian analyses and read into MESQUITE; variation in pod types was examined by ancestral state reconstruction using MESQUITE, version 2.72 (Maddison & Maddison, 2009). Ancestral state reconstruction of characters was carried out using maximum parsimony and maximum likelihood methods. A probability of more than 0.60 for a character compiled from the 100 most probable trees at each node was considered to be the most probable state at that node. RESULTS MATERIAL AND METHODS TAXON SAMPLING Mature pods of 28 species and four varieties representing 32 accessions of Indigofera were collected through our field work carried out in different parts of POD MORPHOLOGY Fruits of Indigofera are generally linear but pods may be flat, compressed (Indigofera hochstetteri), curved [Indigofera nummulariifolia (L.) Livera ex Alston, Indigofera constricta (Thwaites) Trimen, © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 176, 260–276 Species number Species names 1 Indigofera aspalathoides Vahl ex DC. 2 Indigofera astragalina DC. 3 Indigofera atropurpurea Buch.-Ham. ex Hornem Indigofera barberi Gamble Indigofera cassioides Rottler ex DC. Indigofera colutea (Burm.f.) Merr. 4 5 6 Voucher Locality Trichomes Chauhan 1468 Madurai Kamraj Pubescent University Campus, Tamil Nadu Chauhan & Tamil Nadu Pubescent AKPandey 1448 Pericarp thickness (μm) Exocarp Endocarp Dehiscent/ indehiscent pod Pod length Pod (mm) types Epidermis single-layered, thick-walled Single-layered, 4–5 layers, collenchymatous, thick-walled vasculature present collenchymatous 4–6 layers, Dehiscent pod 13.18 sclerenchymatous Type II 384.75 Epidermis single-layered, thick-walled 6–9 layers, Dehiscent pod 13.68 sclerenchymatous Type III 463.12 Epidermis single-layered, thick Walled 10–12 layers, Dehiscent pod 40.05 sclerenchymatous Type III Chauhan 1472 Megamalai hills, Glabrous 171 Madurai, Tamil Nadu Chauhan 1474 Megamalai hills, Glabrous 328.89 Madurai, Tamil Nadu Chauhan 1479 Madurai Kamraj Multicellular as 123.5 University well Campus, unicellular Tamil Nadu Epidermis single-layered, thick-walled Epidermis single-layered, thick-walled Epidermis single-layered, thick-walled 3–5 layers, 1st section contains 4–7 thick-walled layers, collenchymatous collenchymatous tissue, 2nd section contains single-layered, large parenchymatous cells 2–3 layers, 3–4 layers, narrow thick-walled, collenchymatous cells, collenchymatous mostly idioblasts are present Single-layered, 4–5 layers, collenchymatous collenchymatous and parenchymatous, some cells are idioblasts 6–8 layers, collenchymatous, some cells are idioblasts 6–8 layers, Dehiscent pod 10.46 sclerenchymatous Type II 6–9 layers, ? sclerenchymatous Type III Chauhan & AKPandey 5019 Rajghad, Sirmaur, H.P. Glabrous Masai plateau, Kohlapur, Maharashtra Pubescent 337.25 Epidermis single-layered, thick-walled 8 Indigofera dosua Buch.-Ham. ex D.Don Chauhan & AKPandey 5003 Sanghra, Sirmaur, H.P. Glabrous 361 Epidermis single-layered, thick-walled 9 Indigofera exilis Grierson & Long Indigofera gangetica Sanjappa Chauhan & AKPandey 5029 Chauhan & AKPandey 5030 Shimla, H.P. Glabrous 248.04 Shimla, H.P. Pubescent 192.09 Epidermis single-layered, thick-walled Epidermis single-layered, thin-walled Indigofera glabra L. Chauhan & AKPandey 5098 Thummallapalli Uranium mining area, Anantapur Dist., Andhra Pradesh Glabrous 180.5 Epidermis double-layered, thick-walled Single-layered, 2–3 layers, narrow elongated collenchymatous collenchymatous and parenchymatous layer, most cells are idioblasts, vaculature present 2–3 layers, 8–10 layers collenchymatous narrow tissue, vasculature present elongated collenchymatous cells, most cells are idioblasts 1st section contains 3–4 layers, collenchymatous tissue, 2nd section contains 3–5 layers, elongated, narrow collenchymatous cells, most of them are idioblasts, vascular bundles are present 2–3 layers, 4–6 layers, collenchymatous, collenchymatous narrow, elongated cells 1st section contains 3–5 layers, collenchymatous, some cells are idioblasts, 2nd section contains 1–3 layers, narrow elongated parenchymatous tissue, most cells are idioblasts. In this region vascular bundles are present. 3–4 layers, narrow cells, collenchymatous, mostly idioblasts are present, vascular bundles are present 26.54 4–6 layers, Dehiscent pod 18.29 sclerenchymatous Type II 8–11 layers, Dehiscent pod 23.2 sclerenchymatous Type III 8–10 layers, sclerenchyma Dehiscent pod 22.52 Type III Up to 8, sclernchymatous Dehiscent pod 30.56 Type II 7–8 layers, Dehiscent pod 21.65 sclerenchymatous tissue Type II 5–7 layers Dehiscent pod 25.44 sclerenchymatous tissue Type II 263 Indigofera dalzellii Chauhan & AKPandey Cooke 1431 11 Mesocarp 212.51 7 10 Hypodermis POD STRUCTURE AND EVOLUTION IN INDIGOFERA © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 176, 260–276 Table 1. Plant accessions used for the anatomical study of Indigofera and detailed anatomical description of each plant 264 Table 1. Continued 12 © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 176, 260–276 13 14 Indigofera glandulosa Wendl. var. sykesii Griffith ex Baker Indigofera hebepetala Benth. ex Baker var. glabra Ali Voucher Locality Trichomes Pericarp thickness (μm) Exocarp Chauhan & AKPandey 1442 Belgaon, Karnataka Pubescent 160.31 Chauhan 5036 Shimla, H.P. Glabrous Chauhan & AKPandey 5033 Badalta, Sirmaur, H.P. Chauhan & AKPandey 5014 Nohradhar, Sirmaur, H.P. Endocarp Epidermis single-layered, thin-walled 4–6 layers, collenchymatous and parenchymatous, narrow, elongated cells, most cells are idioblasts 4–6 layers, Delayed sclerenchymatous dehiscent 392.63 Epidermis single-layered, thick-walled 8–10 layers, sclerenchyma Glabrous 301 Epidermis single-layered, thick-walled 1st section contains 1–2 layers, rounded collenchymatous cells, 2nd section contains 1–2 layers, rounded collenchymatous cells, most cells are idioblasts, 3rd section contains 4–5 layers, narrow elongated parenchymatous cells, vascular bundles present 6–10 layers, collenchymatous, most cells are idioblasts Glabrous 508.91 Epidermis single-layered, thick-walled Epidermis single-layered, thick-walled Epidermis single-layered, thick-walled Chauhan 5032 Budgaam, J & K Glabrous 318.25 Chauhan & AKPandey 6002 Hissar Dist., Punjab Pubescent 47.5 18 Indigofera karnatakana Sanjappa Chauhan & AKPandey 5046 Bannerghatta hills, Karnataka Glabrous 228 Epidermis single-layered, thick-walled 19 Indigofera karuppiana Pallithanam Chauhan 1465 Kannalti, Niligris Dist., Tamil Nadu Pubescent 184.3 Epidermis single-layered, thick-walled 20 Indigofera linifolia (L. f.) Retz. var. campbelli Wight ex Baker Chauhan & AKPandey 1446 Pubescent 167.77 Epidermis single-layered, thick-walled 21 Indigofera linifolia (L. f.) Retz. var. linifolia Indigofera longiracemosa Boivin ex Baill. Chauhan & AKPandey 1470 Madurai Kamraj Pubescent University Campus, Tamil Nadu Chauhan 1498 Kalkadu, Tirunelveli Dist., Tamil Nadu 202.35 Epidermis single-layered, thick-walled 15 16 17 22 University Campus Garden, Kohlapur, Maharashtra Dehiscent/ indehiscent pod Mesocarp Indigofera hebepetala Benth. ex Baker var. hebepetala Indigofera heterantha Wallich ex Brandis Indigofera himalayensis Ali Indigofera hochstetteri Baker 352.45 Hypodermis 4–5 layers, 4–8 layers, narrow elongated thick-walled collenchymatous and collenchymatous parenchymatous, most cells are idioblasts 1–2 layers, ? thick-walled collenchymatous 1st section contains 2–4 layers, collenchymatous, most cells are idioblasts, vasculature present Double-layered 1st section contains 2–3 thick-walled layers, parenchymatous, collenchymatous 2nd section contains 3–4 layers narrow, collenchymatous most cells are idioblasts Single-layered 1st section contains 4–5 thick-walled layers, narrow, elongated collenchymatous parenchymatous tissue, most cells are idioblasts Single-layered, 1st section contains 2–3 thick-walled layers, narrow elongated collenchymatous collenchymatous cells, most cells are idioblats, vasculature is present in this layer, 2nd section contains single-layered rounded collenchymatous cells 1–2 layers, 1–2 layers, collenchymatous, collenchymatous some cells are idioblasts Pod length Pod (mm) types 2.49 Type II Dehiscent pod 50.45 Type III 11–14 layers, Dehiscent pod 50.04 sclerenchymatous Type III 8–10 layers, sclerenchyma 31.86 Type III 8–10 ? sclerenchymatous 4.02 Type III 3–5 layers, Delayed sclerenchymatous dehiscent 11.56 Type I 4–6 layers, Delayed sclerenchymatous dehiscent 22.5 Type II Dehiscent pod 7.42 Type II 6–7 layers, sclerenchyma ? 3–6 layers, Dehiscent pod 2.1 sclerenchymatous Type II 2–6 layers, Dehiscent pod 2.1 sclerenchymatous Type II 7–9 layers, sclerenchymatous Type III 15.35 V. CHAUHAN and A. K. PANDEY Species number Species names Indigofera oblongifolia Forssk. Chauhan & AKPandey 5099 24 Indigofera prostrata Willd. Chauhan & AKPandey 1451 25 Indigofera sessiliflora DC. Chauhan& AKPandey 6001 26 Indigofera tinctoria L. 27 Indigofera trifoliata L. 28 Indigofera trifoliata L. var. duthiei (Drumm. ex Naik) Sanjappa Indigofera trita L. f. var. scabra (Roth) Ali Indigofera trita L. f. var. trita 29 30 31 32 Indigofera uniflora Buch.-Ham. ex Roxb. Indigofera wightii Graham ex Wight & Arn. Thummallapalli Uranium mining area, Anantapur Dist., Andhra Pradesh Goa University Campus, Goa Ramgard, Jaisalmer Dist., Rajasthan Chauhan 1467 Kannalti, Tamil Nadu Chauhan & AKPandey 1438 Chauhan & AKPandey 1429 Sutghatti ghat, Kohlapur, Maharashtra Girgaon, Kohlapur, Maharashtra Chauhan 1460 Marudhamalai hills, Tamil Nadu Chauhan 1471 American College, Tamil Nadu Chauhan 1459 Madhukkarai hills, Tamil Nadu Chauhan 1473 Megamalai hills, Madurai, Tamil Nadu Pubescent 155.13 Epidermis single-layered, thick-walled Sparsely pubecent 129.77 Epidermis single-layered, thick-walled Pubescent 194.18 Epidermis single-layered, thick-walled Glabrous 215.65 Epidermis single-layered, thick-walled Pubescent 181.45 Pubescent 123.5 Epidermis single-layered, thick-walled Epidermis single-layered, thick-walled Pubescent 181.64 Pubescent 224.39 Pubescent 95.95 Pubescent 396.15 Epidermis single-layered, thick-walled Epidermis single-layered, thick-walled Epidermis single-layered, thick-walled Epidermis single-layered, thick-walled 4–8 layers, collenchymatous 3–6 layers, Dehiscent pod 12.42 sclerenchymatous Type II Single-layered, 1–2 layers, narrow, collenchymatous elongated, collenchymatous and parenchymatous, most cells are idioblasts, vascular bundles present 1–2 layers, 4–6 layers, collenchymatous thick-walled, tissue. Vasculature present collenchymatous 4–6 layers, Dehiscent pod 9.72 sclerenchymatous Type II 4–6 layers, Dehiscent pod 7.86 sclerenchymatous Type II 1st section contains 2–3 layers, collenchymatous tissue, 2nd section contains 2–4 layers, narrow large elongated parenchymatous, most cells are idioblasts Single-layered, 3–4 layers, narrow elongated thick-walled, collenchymatous, most collenchymatous cells are idioblasts Single-layered, 3–4 layers, collenchymatous, thick-walled, some cells are idioblasts collenchymatous 6–8 layers, Dehiscent pod 23.9 sclerenchymatous Type II 6–8 layers, Dehiscent pod 7.48 sclerenchymatous Type II 4–6 layers, ? sclerenchymatous 7.76 Type II 1–2 layers, 6–8 layers, narrow elongated thick-walled, collenchymatous, collenchymatous vasculature present 1–2 layers, 4–5 layers, collenchymatous thick-walled, and parenchymatous cells collenchymatous 3 layers, collenchymatous 4–6 layers, Delayed sclerenchymatous dehiscent 35.67 Type II 5–7 layers, Dehiscent pod 24.84 sclerenchymatous tissue 4–5 layers, ? 6.84 sclerenchymatous Type II 7–9 layers, Dehiscent pod 23.18 sclerenchymatous Type III 1st section conatins 4–6 layers, collenchymatous tissue, 2nd section contains 3–5 layers, elongated large parenchymatous cells Vascular bundles are present Type I POD STRUCTURE AND EVOLUTION IN INDIGOFERA © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 176, 260–276 23 265 266 V. CHAUHAN and A. K. PANDEY Table 2. Character matrix showing the character states for all the species used in ancestral state reconstruction in Mesquite Taxon/character Pod type Taxon/character Pod type Cyamopsis tetragonoloba FJ769262 Indigastrum argyroides EU729488 Indigastrum candidissimum EU729489 Indigastrum costatum AF521716 Indigastrum fastigiatum EU729490 Indigofera dosua AF534790 Indigofera himalayensis AF534792 Indigofera aspalathoides JQ945958 Indigofera astragalina EU729545 Indigofera astragalina JQ945966 Indigofera atropurpurea EU729575 Indigofera barberi JQ945961 Indigofera bongardiana EU729661 Indigofera cassioides EU729573 Indigofera cassioides JQ945965 Indigofera colutea AF521776 Indigofera colutea JQ945956 Indigofera concava EU729619 Indigofera cordifolia AF521741 Indigofera cordifolia JQ945963 Indigofera cuneifolia AF521749 Indigofera dalzellii AF521793 Indigofera dalzellii JQ945962 Indigofera denudata EU729617 Indigofera dosua AF521771 Indigofera drepanocarpa EU729632 Indigofera gairdnerae EU729507 Indigofera glabra EU729504 Indigofera glandulosa EU729634 Indigofera glandulosa var. skyseii JQ945967 Indigofera hebepetala AF521768 Indigofera hebepetala AF534793 2 2 2 2 2 2 2 1 2 2 2 1 ? 2 2 1 1 ? ? ? ? 2 2 ? 2 ? ? 1 1 1 2 2 Indigofera Indigofera Indigofera Indigofera Indigofera Indigofera Indigofera Indigofera Indigofera Indigofera Indigofera Indigofera Indigofera Indigofera Indigofera Indigofera Indigofera Indigofera Indigofera Indigofera Indigofera Indigofera Indigofera Indigofera Indigofera Indigofera Indigofera Indigofera Indigofera Indigofera Indigofera Indigofera 2 ? 2 0 0 1 1 1 1 ? ? 2 2 1 ? ? 1 1 ? ? 1 1 ? 1 1 1 1 1 1 0 2 2 heterantha EU729571 heterotricha EU729535 himalayensis AF521770 hirsuta EU729546 hochstetteri EU729656 karnatakana EU729544 lespedezioides AF521780 linifolia EU729629 linifolia JQ945964 linnaei EU729670 linnaei JQ945960 longiracemosa AF521786 longiracemosa AY124764 microcarpa EU729630 miniata EU729663 nummulariifolia EU729631 oblongifolia AF521778 prostrata JQ945972 schimperi AF274696 semitrijuga EU729669 sessiliflora EU729642 spicata EU729671 squalida EU729633 suffruticosa AF467051 tinctoria AF521775 trifoliata JQ945971 trita AF521745 trita EU729681 truxillensis EU729602 uniflora JQ945959 wightii EU729519 wightii JQ945957 Coding for the character states – pod types: 0, pod type I; 1, pod type II; 2, pod type III. Indigofera suffruticosa Mill., and Indigofera trita var. scabra] or globose [Indigofera cordifolia Heyne ex Roth, Indigofera linifolia (Fig. 2). The cross-section of the pod is usually cylindric, subcylindric or subtetragonous, except Indigofera nummulariifolia in which it is triquetrous. The pods are usually adpressed hairy, except in some species where they are glabrous (Indigofera atropurpurea, Indigofera cassioides, Indigofera cedrorum Dunn, Indigofera cylindracea Graham ex Baker, Indigofera exilis, Indigofera glabra, Indigofera hamiltonii Graham ex Duthie & Prain, Indigofera hebepetala, Indigofera himalayensis, Indigofera lacei Craib). The pod is deflexed or spreading or ascending, dehiscent or indehiscent. In some species (Indigofera barberi, Indigofera glandulosa, Indigofera karuppiana, Indigofera pedicellata Wight & Arn., Indigofera prostrata, Indigofera santapaui Sanjappa, Indigofera thothathrii Sanjappa, Indigofera tirunelvelica Sanjappa), pods have glands on the surface. All the species having gland dotted pods are trifoliolate species. Multicellular, erect, gland-tipped hairs are present on pods in Indigofera argentea and Indigofera colutea. In some species of Indigofera, mature fruits have broad sutures (Indigofera argentea Burm.f., Indigofera arrecta Hochst. ex A.Rich, Indigofera bracteata Graham ex Baker, I. colutea, Indigofera dosua, I. suffruticosa, Indigofera tirunelvelica, Indigofera zollingeriana Miq.), fruits with obscurely or shortly winged sutures [Indigofera dalzellii, Indigofera glabra, Indigofera karuppiana, Indigofera prostrata, Indigofera thothathrii, Indigofera trifoliata var. duthiei], fruits with prominently winged sutures (Indigofera bracteata var. khasiana Sanjappa, Indigofera © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 176, 260–276 POD STRUCTURE AND EVOLUTION IN INDIGOFERA 267 Figure 2. Morphological variation of fruits of genus Indigofera. A. Indigofera trita var. scabra (1460). B, Indigofera wightii (1473). C, Indigofera cordifolia (1432). D, Indigofera linifolia (1470). E, Indigofera linnaei (1469). F, Indigofera cassioides (1474). G, Indigofera karuppiana (1465). H, Indigofera hendecaphylla (1477). I, Indigofera trifoliata (1438). J, Indigofera trita var. trita (1471). K, Indigofera astragalina (1448). L, Indigofera dosua (1488). M, Indigofera tinctoria (1475). N, Indigofera heterantha (1485). O, Indigofera colutea (1479). P, Indigofera dalzellii (1431). Q, Indigofera aspalathoides (1468). R, Indigofera prostrata (1451). S, Indigofera glandulosa (1442). T, Indigofera barberi (1472). U, Indigofera trifoliata var. duthiei (1429). V, Indigofera uniflora (1459). W, Indigofera himachalensis (1487). Scale bar = 2 mm. glandulosa, Indigofera trifoliata) or spiny processes along the sutures (Indigofera nummulariifolia). POD ANATOMY Fruits in Indigofera are relatively thin- to thickwalled. Anatomically, the pericarp is distinguishable into exocarp, mesocarp and endocarp. Exocarp The exocarp generally consists of a single-layered epidermis, except in I. glabra where it is biseriate. The exocarp is mostly thick-walled, except Indigofera gangetica and I. glandulosa var. sykesii, in which it is thin-walled. The hypodermis may be present or absent. If present, then it is usually single-layered, but sometimes multiple layers are present (Indigo- © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 176, 260–276 268 V. CHAUHAN and A. K. PANDEY fera astragalina, I. atropurpurea, Indigofera dalzellii, I. exilis, Indigofera heterantha and Indigofera karnatakana). The multilayered exocarp is composed of thick-walled collenchymatous cells. Mesocarp The mesocarp is composed of three or four to eight- to ten-layered collenchymatous tissue but, in some species, parenchymatous tissue is also present (e.g., Indigofera astragalina, Indigofera barberi, Indigofera colutea, Indigofera gangetica, Indigofera glandulosa var. sykesii, Indigofera hebepetala var. glabra, Indigofera heterantha, Indigofera karnatakana, Indigofera karuppiana, Indigofera prostrata, Indigofera tinctoria, Indigofera trita var. trita, Indigofera wightii). Vascular bundles and most of the idioblasts are present in the mesocarp region. Endocarp The endocarp is composed of a variable number of sclerenchyma layers, ranging from three to five to ten to 12. The most common endocarp cell layer number in Indigofera is six to eight. On the basis of pericarp thickness and number of sclerenchymatous layers in the endocarp, three basic fruit types can be recognized: pod type I, II, and III. Type I (thin pericarp and three to five sclerenchymatous layers in the endocarp); type II (intermediary pericarp thickness and six to eight sclerenchymatous layers); and type III (thick pericarp and more than eight sclerenchymatous layers) (Figs 3, 4, 5; Table 3). The anatomical description for the genus is summarized in Table 4 with an indication of the fruit pericarp type (fruit type classification for all species investigated is listed in the Table 1). The distribution of fruit pericarp types in the tribe is shown in Figure 6. POD DEHISCENCE Both dehiscent and tardily dehiscent pods are present in Indigofera, which generally shows normal explosive dehiscence as a mean of dispersal of seeds, although some species show adaptations for dispersal by wind (I. cordifolia, I. linifolia, I. glandulosa, and I. linnaei Ali). Some Indigofera spp. are similar in having small, light weighted, few seeded, indehiscent or tardily dehiscent fruits with persistent calyces and corollas with the pod embellished with broad sutures, leading to longitudinal fringed wings, appearing to be adapted to dispersal by wind, water or gravity. All these fruit characters indicate a shift to pods playing a role in seed dispersal. The whole pod acts as a diaspore that can easily blown away by wind. In Indigofera, pods usually have separation tissue present along the sutures and a variable number of sclerenchymatous layers in the endocarp. These two characteristics are indicative of a dehiscent pod. However, in some taxa (I. hochstetteri, I. karnatakana, I. glandulosa var. sykesii, and I. trita var. scabra), which show a tardily dehiscent or indehiscent condition, no separation tissue is found and there is mostly a low to intermediate number of sclerenchyma layers present in the endocarp. Two Neotropical Indigofera spp., I. microcarpa Desv. (winged pods) and I. suffruticosa (broad sutures but no wings), are tardily dehiscent because there is no separation tissue present (Leite et al., 2009). Roth (1977) reported two anatomical features that appear to reduce the tendency to dehisce in legumes. The first is a longer fruit and the second is a strongly bent fruit. The indehiscent I. trita var. scabra possesses a long and strongly curved pod. EVOLUTION OF POD ANATOMICAL CHARACTERS The evolutionary pattern of pod types was inferred from ancestral state reconstruction (Fig. 6), carried out on the 100 most probable trees from Bayesian analyses (TRPROBS file) and the probabilities of a particular ancestral state at specific nodes (Table 5). In the ancestor to Cyamopsis DC. + Indigastrum Jaub. & Spach + Indigofera, however, the two states pod type II and III are present but pod type III probability (P = 0.6245) is higher than for other states. In Indigastrum and Cyamopsis, the pod is of type III. Pod type II was present (P = 0.7471) in ancestral Indigofera. The common ancestors of clades C (P = 0.7311), D (P = 0.9784), and E (P = 0.9542) have pod type II present, whereas the ancestor of clade F had pod type III (P = 0.6033). The transition from pod type II to pod type III takes place at node f. There is a reversal in I. wightii (clade E) and I. dalzellii (clade C), which have pod type III. In I. karnatakana and I. tinctoria–I. truxillensis Kunth– I. suffruticosa (clade F), pod type II has evolved independently from the pod type III condition. In Indigofera uniflora (clade E) and I. hochstetteri (clade C), pod type I has evolved independently from the pod type II condition and, simultaneously, in Indigofera hirsuta L. (clade F), pod type I has evolved independently from pod type III. DISCUSSION In Indigofera, pod morphology is variable and characters have little diagnostic value at the generic level. The genus can be identified by a combination of characters, and fruit characters on their own have limited value (Gillett, 1958; De Kort & Thijsse, 1984; Sanjappa, 1995; Schrire, 1995; Wilson & Rowe, 2004, 2008). An example of nonhomologous similarity was © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 176, 260–276 POD STRUCTURE AND EVOLUTION IN INDIGOFERA 269 Figure 3. Transverse sections of fruits in Indigofera. Paleotropical clade with a type I pericarp (A), type II pericarp (B, C, D, E), and type III pericarp (F). A, Indigofera uniflora [Chauhan 1459]. B, Indigofera glabra [Chauhan & AKPandey 5098]. C, Indigofera colutea [Chauhan 1479]. D, Indigofera aspalathoides [Chauhan 1468]. E, Indigofera barberi [Chauhan 1472]. F, Indigofera wightii [Chauhan 1473]. Scale bar = 50 μm. revealed by the presence of flattened and compressed pods that are found in Indigofera, Cyamopsis, and Indigastrum. In Cyamopsis tetragonoloba Taub., fruits are longitudinally ridged and septate between seeds. The anatomy of the pericarp is too conservative to be used diagnostically at the generic level. In Indigofera glabra, the exocarp is biseriate, a character also present in the Neotropical species Indigofera lespedezioides Kunth (Leite et al., 2009). The exocarp is mostly thin-walled in Neotropical species [Indigofera campestris Bong. ex Benth., I. hirsuta, I. lespedezioides, I. spicata Forssk. (introduced to the New World), I. suffruticosa and I. truxillensis (Leite et al., 2009)]. However, some trends were observed by us and three basic pericarp types were identified within the tribe. The distribution of these types across the tribe is not congruent with current phylogenetic analyses, as shown in Figure 6. Our anatomical studies on the Indigofera spp. reveal that the genus is closely related to Cyamopsis– Indigastrum clade of Schrire et al. (2003, 2009). All the species that were sampled for the study belong to four clades (Palaeotropical, Pantropical, Cape, and Tethyan clades of Indigofera as given by Schrire et al., 2003, 2009). The whole Palaeotropical–Pantropical, Cape, and Tethyan clades form polytomy with each other. Ancestral state reconstructions of pod type © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 176, 260–276 270 V. CHAUHAN and A. K. PANDEY Figure 4. Transverse sections of fruits in Indigofera. Pantropical clade with a type II pericarp (A, B), and type III pericarp (C, D, E, F, G, H). A, Indigofera karnatakana [Chauhan & AKPandey 5046]. B, Indigofera tinctoria [Chauhan 1467]. C, Indigofera astragalina [Chauhan & AKPandey 1448]. D, Indigofera heterantha [Chauhan & AKPandey 5014]. E, Indigofera hebepetala var. hebepetala [Chauhan & AKPandey 5033]. F, Indigofera hebepetala var. glabra [Chauhan 5036]. G, Indigofera dosua [Chauhan & AKPandey 5003]. H, Indigofera himalayensis [Chauhan 5032]. Scale bar = 50 μm. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 176, 260–276 POD STRUCTURE AND EVOLUTION IN INDIGOFERA 271 Figure 5. Transverse sections of fruits in Indigofera: Tethyan clade with a type I pericarp (A), type II pericarp (B, C, D, E, F, G) and type III pericarp (H). A, Indigofera hochstetteri [Chauhan & AKPandey 6002]. B, Indigofera trifoliata [Chauhan & AKPandey 1438]. C, Indigofera prostrata [Chauhan & AKPandey 1451]. D, Indigofera glandulosa var. sykesii [Chauhan & AKPandey 1442]. E, Indigofera trita var. trita [Chauhan 1471]. F, Indigofera linifolia [Chauhan & AKPandey 1446]. G, Indigofera sessiliflora [Chauhan& AKPandey 6001]. H, Indigofera dalzellii [Chauhan & AKPandey 1431]. Scale bar = 50 μm. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 176, 260–276 272 V. CHAUHAN and A. K. PANDEY Table 3. Characteristics of the three basic fruit types recognized in the genus Indigofera Pod type Pericarp Sclerenchymatous thickness layers Fruit structure Type I Below Three to 100 μm five layers Type II 100– Six to eight 300 μm layers Type III Above More than 300 μm eight Distribution in the genus Present in Indigofera uniflora and Fruits relatively thin-walled, trichomes Indigofera hochstetteri. present. Exocarp: single-layered epidermis, thick- walled. Hypodermis absent. Mesocarp: only collenchymatous cells. Endocarp: three- to five layered, sclerenchymatous. Delayed dehiscent pod (Figs 3, 5) Predominantly present in Fruits are of intermediatary thickness, Indigofera spp. trichomes absent/present. Exocarp: oneor or two-layered epidermis, thin to thick-walled. Hypodermis absent/present, single-layered to two- to three-layered thick-walled collenchymatous. Mesocarp: composed of collenchymatous cells or both collenchymatous and parenchymatous cells. Endocarp: six- to eight-layered, sclerenchymatous. Dehiscent or delayed dehiscent pod (Figs 3, 4, 5) Present in Indigofera astragalina, Fruits relatively thick-walled, trichomes Indigofera atropurpurea, absent/present. Exocarp: epidermis Indigofera cassioides, Indigofera single-layered, thick- walled. Hypodermis dalzellii, Indigofera dosua, absent/present, single to four- to Indigofera hebepetala, Indigofera five-layered thick-walled collenchymatous. heterantha, Indigofera Mesocarp: comprises collenchymatous himalayensis, Indigofera cells or both collenchymatous and longiracemosa, and Indigofera parenchymatous cells. Endocarp: eight- to wightii 11-layered sclerenchymatous. Dehiscent pod (Figs 3, 4, 5) show that the common ancestor of tribe Indigoferae has pod type III. The common ancestor of Indigofera had pod type II (P = 0.7471) and a change occurred in the ancestor of clade F to pod type III (P = 0.6033). Species of Indigastrum and Cyamopsis have a type III pericarp, whereas, in Indigofera, all three types (I, II, III) are present. Type II is predominant and occurs in the Palaeotropical and Tethyan clades, and independently evolved in I. karnatakana, I.truxillensis, I. suffruticosa, and I. tinctoria; type I occurs in three independent lineages (I. uniflora, I. hirsuta, and I. hochstetteri) and type III is observed in Indigastrum, Cyamopsis, and the pantropical clade with an independent reversal to type III in I. wightii and I. dalzellii. The three parameters of the pod (pod size, pericarp thickness, and number of sclerenchyma layers in the endocarp) are correlated. There is a correlation between the size of the pod (length) and the thickness of the endocarp (sclerenchyma tissue) because all the large pods (> 20 mm) have a thick endocarp, small pods (< 10 mm) have a thinner endocarp and inter- mediate pods (10–20 mm) has intermediate endocarp (e.g. I. karuppiana, I. glabra, I. trifoliata, I. trita var. trita). Pericarp thickness is directly correlated with number of sclerenchymatous layers in the endocarp. Molecular and morphological data (Schrire et al., 2009) indicated that Indigastrum parviflorum (B.Heyne ex Wight & Arn.) Schrire should be excluded from Indigofera, although its placement in Indigastrum is not supported by fruit morphology or anatomy (V. Chauhan & A. K. Pandey, unpubl. data). The type III pericarp (present in the early diverging lineages of the tribe) represents the least derived state, whereas type I and II are more derived. Fahn & Zohary (1955) considered the presence of multiple cell layers of sclerenchyma in the endocarp to be the primitive state in legumes and its loss to be derived. Our results also support the earlier findings of Fahn & Zohary (1955), indicating that the small pod, thin pericarp, and fewer sclerenchymatous layers in the endocarp (three to five) (pod type I) and the intermediate pod condition and thin- to thick-walled and five to eight sclerenchymatous layers in the endocarp (pod type II) are more © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 176, 260–276 Pericarp thickness in micrometers Exocarp Species names Indigofera aspalathoides Vahl ex DC. Indigofera astragalina DC. Indigofera atropurpurea Buch.-Ham. ex Hornem Indigofera barberi Gamble Indigofera cassioides Rottler ex DC. Indigofera colutea (Burm.f.) Merr. Indigofera dalzellii T.Cooke Indigofera dosua Buch.-Ham. ex D.Don Indigofera exilis Grierson & Long Indigofera gangetica Sanjappa Indigofera glabra L. Indigofera glandulosa Wendl. var. sykesii Griffith ex Baker Indigofera hebepetala Benth. ex Baker var. glabra Ali Indigofera hebepetala Benth. ex Baker var. hebepetala Indigofera heterantha Wallich ex Brandis Indigofera himalayensis Ali Indigofera hochstetteri Baker Indigofera karnatakana Sanjappa Indigofera karuppiana Pallithanam Indigofera linifolia (L. f.) Retz. var. campbelli Wight ex Baker Indigofera linifolia (L. f.) Retz. var. linifolia Indigofera longiracemosa Boivin ex Baill. Indigofera oblongifolia Forssk. Indigofera prostrata Willd. Indigofera sessiliflora DC. Indigofera tinctoria L. Indigofera trifoliata L. Indigofera trifoliata L. var. duthiei (Drumm. ex Naik) Sanjappa Indigofera trita L. f. var. scabra (Roth) Ali Indigofera trita L. f. var. trita Indigofera uniflora Buch.-Ham. ex Roxb. Indigofera wightii Graham ex Wight & Arn. Hypodermis Mesocarp Endocarp Dehiscence Pod types + < 100 μm ++ 100–300 μm +++ > 300 μm Number of Cell walls _absent exocarp -thin +present layers +thick + 3–5 layers, thin ++ 6–8 layers, Parenchyma Collenchyma intermediate +Dehiscent pod –absent –absent +++ > 8 layers, –Delayed +present +present thick dehiscent pod Type I/II/III ++ +++ +++ ++ +++ ++ +++ +++ ++ ++ ++ ++ One One One One One One One One One One Two One + + + + + + + + + _ + _ + + + + _ + + _ + _ _ _ _ + _ + _ + _ _ _ + _ + + + + + + + + + + + + + ++ +++ +++ ++ +++ ++ +++ +++ ++ ++ ++ ++ + + + + ? + + + + + + _ Type Type Type Type Type Type Type Type Type Type Type Type II III III II III II III III II II II II +++ +++ +++ +++ + ++ ++ ++ One One One One One One One One + + + + + + + + _ _ + + _ + + + + _ + + + + _ + + _ + + _ + +++ +++ +++ +++ + ++ ++ ++ + + ? ? _ _ + + Type Type Type Type Type Type Type Type III III III III I II II II ++ +++ ++ ++ ++ ++ ++ ++ One + + _ + One One One One One One + + + + + + + + + _ + + _ + _ + _ _ + + + + + + ++ +++ ++ ++ ++ ++ ++ ++ + ? + + + + + ? Type Type Type Type Type Type Type Type II III II II II II II II ++ ++ + +++ One One One One + + + + + _ _ _ _ + _ + + + + + ++ ++ + +++ _ + ? + Type Type Type Type II II I III 273 Type I, below 100 μm and 3–5 sclerenchymatous layers; type I, 100–300 μm and 3–8 layers; type II, above 300 μm and > 8 layers. POD STRUCTURE AND EVOLUTION IN INDIGOFERA © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 176, 260–276 Table 4. Summary of pericarp characters of genus Indigofera 274 V. CHAUHAN and A. K. PANDEY Figure 6. Phylogram constructed from 1998 trees in MrBayes. All the clades are labelled. Numbers on the branches indicate the posterior probabilities (pp). All the coloured branches correspond to the four clades formed by Schrire et al., 2009. A black line shows the evolution of the pod type character at specific nodes carried out from the ancestral state reconstruction by parsimony and likelihood methods in MESQUITE. The likelihoods were calculated for the 100 most probable trees in the TRPROBS file in MrBayes. The character states marked show the most probable states at that node. Table 5. Summary of evolution of pod types in tribe Indigoferae Pod types Pod type I Pod type II Pod type III A – Recent common ancestor to Cyamposis + Indigastrum + Indigofera clade B – Ancestral Indigofera C – Ancestral Palaeotropical + Pantropical clade D – Ancestral Tethyan clade E – Ancestral Palaeotropical clade F – Ancestral Pantropical clade 0.0287652 in 100 trees 0.3467892 in 100 trees 0.624524 in 100 trees 0.0077725 in 100 trees 0.006239 in 100 trees 0.747155 in 100 trees 0.731111 in 100 trees 0.24512 in 100 trees 0.262650 in 100 trees 0.00397931 in 100 trees 0.0016906 in 100 trees 0.97843279 in 100 trees 0.9542524 in 100 trees 0.0175879 in 100 trees 0.044057 in 100 trees 0.0049752 in 100 trees 0.3916416 in 100 trees 0.6033832 in 100 trees Maximum likelihood values for reconstruction of pod types (Pod type I, II, III) at specific nodes of the tree (Fig. 6) taken from the 100 most probable trees from the TRPROBS file retrieved from MrBayes. Probabilities which are more than 0.6 are in bold because they are considered to be the most probable state for that node. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 176, 260–276 POD STRUCTURE AND EVOLUTION IN INDIGOFERA derived compared to the large pod type with thick pericarp and more sclerenchymatous layers in the endocarp (more than eight) (pod type III). It is evident from character reconstruction that an increase in pericarp thickness and number of sclerenchyma layers (pod type III) is probably a plesiomorphic trait of the genus and a decrease in pericarp thickness and number of sclerenchyma layers (pod type II and pod type I) is apomorphic for the tribe. In Indigofera, dehiscence is caused by a separation layer present at the dorsal and ventral sutures except in some species (I. hochstetteri, I. karnatakana, I. glandulosa var. sykesii, I. trita var. scabra) where no separation tissue is present and the species show a tardily dehiscent or an indehiscent condition. The indehiscent pod type is considered to be apomorphic. ACKNOWLEDGEMENTS Grant support comprising an R&D Grant of the University of Delhi to AKP is thankfully acknowledged. REFERENCES Buckovic RG. 1952. Some of the morphological and agronomic factors associated with pod dehiscence in Lotus corniculatus. MSc Thesis, Oregon State University. Chauhan V, Pandey AK, Schaefer H. 2013. Indigofera himachalensis (Fabaceae: Indigofereae), a new species from Himachal Pradesh, India. Phytotaxa 112: 43–49. Dave Y, Bennet R. 1990. The complete architecture of fruit Cyamopsis tetragonoloba (L.) DC. Journal of Phytological Research 3: 13–21. De Kort I, Thijsse G. 1984. 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