Egypt. J. Bot. Vol. 59, No.2, pp. 475 - 482 (2019) 35 Dicliptera aegyptiaca (Acanthaceae), A New Species from Egypt Supported by Morphological Characters and rbcl-based DNA Barcoding Eman M. Shamso(1), Ahmed S. Fouad(2)# The Herbarium, Botany and Microbiology Department, Faculty of Science, Cairo University, Giza 12613, Egypt; (2)Botany and Microbiology Department, Faculty of Science, Cairo University, Giza 12613, Egypt. (1) D ICLIPTERA aegyptiaca, a new species from Red Sea Coast, Egypt, is described and illustrated. Diagnostic and morphological characters that distinguish it from its allied species D. paniculata and an identification key for the two species are provided. The new species differs from D. paniculata by having an unbranched stem, a congested inflorescence with dwarf axes 1.5– 5mm long; subsessile cymules with peduncles 0.5– 1mm long. rbcl-DNA barcoding is presented for this new taxon for the first time. Phylogentic tree revealed barcode clusters for the two Dicliptera species and recognized significant interspecific variation between them. D. aegyptiaca clearly formed one clade strongly supported with a bootstrap value of 100%. Based on characters of morphology, pollen and seeds, the new species was recognized as belonging to the genus Dicliptera. On the other hand, DNA barcoding reflected clustering of all Dicliptera spp. in a large clade while D. aegyptiaca formed a non sister clade showing the utility of DNA barcoding for species identification rather than taxonomy. Keywords: Dicliptera aegyptiaca, DNA barcoding, Egypt, Morphology, New species. Introduction Peristrophe are together monophyletic. The genus Dicliptera Juss. comprises about 175 species (Daniel, 2009), distributed in tropical and subtropical countries of Asia, Africa and America. Within Acanthaceae, it is placed in the Diclipterinae clade of the “Justicioid” lineage with Hypoestes as a sister to Peristrophe (McDade et al., 2000 a). In the absence of fruits, it is difficult to distinguish between Peristrophe and Dicliptera s. str. (Balkwill, 1996 and Darbyshire & Vollesen, 2007). Based on the mechanism of capsule dehiscence, Balkwill (1996) and Balkwill et al. (1985, 1986, 1996) maintained that the two taxa should remain separate, where the capsule is elastic in Dicliptera and inelastic in Peristrophe. In their work, Darbyshire & Vollesen (2007) transferred all recognized Peristrophe from Tropical East Africa to Dicliptera citing inconsistencies of capsule types and the presence of intermediate forms. The most recent molecular data on the “Justicioid” lineage, Kiel et al. (2017) support Darbyshire & Vollesen’s treatment as sampled species of Dicliptera and Dicliptera (inclusive of Peristrophe) is characterized by angled stem, opposite leaves, inflorescence panicle-like cymose, with 2–3 inflorescence units (cymules), umbellately arranged, and a resupinate bilabiate corolla. Fruitcapsule clavate, 4-seeded. # In Egypt, the genus Dicliptera was represented by only one species D. paniculata (Peristrophe paniculata), a rare species known only from Gebel Elba and Red Sea Coast of Egypt (Täckholm, 1974; El Hadidi & Fayed, 1994/ 95; Boulos, 2002, 2009; Shamso, 2010, 2013 and El-Gazzar et al., 2015). As part of the revisionary study of the genus Dicliptera in Egypt, Shamso (2010) recorded Dicliptera paniculata with a wide range of variation in many morphological characters. Most examined specimens represented D. paniculata, while some other specimens did not match and were probably an undescribed taxon. Their identities were confirmed by referring to various regional floras Corresponding author email: ahmedsfouad@yahoo.com DOI: 10.21608/ejbo.2019.5371.1220 Edited by: Prof. Dr. Adel El-Gazzar, Faculty of Science, El-Arish University, El-Arish, N. Sinai, Egypt. ©2019 National Information and Documentation Center (NIDOC) 476 EMAN M. SHAMSO AND AHMED S. FOUAD (Clarke, 1901; Andrews, 1956; Hein, 1966; Abedin et al., 2000; Hedrén, 2006; Ensermu Kelbessa, 2006 and Darbyshire et al., 2010, 2015) and much other relevant literatures (Balkwill, 1996; Balkwill & Getliffe Norris, 1989; Balkwill et al., 1985, 1986, 1988, 1996; Ensermu Kelbessa, 2003; Darbyshire & Vollesen, 2007 and Al Hakimi et al., 2017). The specimens were also compared with herbarium specimens and images available in virtual herbaria [Kew Herbarium Catalogue (http://apps.kew.org/ herbcat/navigator.do), African Plant Database (www.tropicos.org) and JSTOR Global Plants (https://plants.jstor.org/)]. It is suggested that these specimens do not match with any of the described species of Dicliptera (incl. Peristrophe) and so are described here as a new species. DNA barcoding provides an accurate, rapid and time-efficient tool for species identification utilizing short DNA sequences as internal species tags (Hebert, 2003 and Hebert & Gregory, 2005). The CBOL plant working group (2009) recommended employing rbcl and/or matk in barcoding of land plants. Compared to matk, rbcl has high amplification success rate; it is the most characterized plastid coding region in GenBank (Newmaster et al., 2006 and Kang et al., 2017). rbcl was used for identification of cryptic species by several research groups (Miwa et al., 2003; Costea & Stefanovic, 2009; Liu et al., 2013 and Ardiyani et al., 2017). The aims of this study are to confirm the occurrence of the new taxon Dicliptera aegyptiaca using macro- and micro-morphological data and DNA barcodes, to provide the validating description of the new taxon and to provide a key to the Egyptian species of Dicliptera for easy recognition of the taxa in Egypt. Materials and Methods Plant materials The present study was based on examination of several specimens belonging to Dicliptera paniculata kept in the major Egyptian herbaria [Cairo University Herbarium (CAI), the Agricultural Research Centre, Flora and Phytotaxonomy Herbarium (CAIM), National Research Centre, Plant Systematic Herbarium (CAIRC) and Sohag University Herbarium (SHG)]. The new taxon is accompanied by English description (ICBN, 2018: article 39.2). Egypt. J. Bot. 59 , No.2 (2019) Pollen and seeds examination Samples of pollen and seeds were taken from mature anthers and capsules, respectively. For light microscopy (LM), anthers were boiled for a few minutes in water, macerated in a few drops of an aqueous 10% solution of KOH on a clean slide, then stained with Safranin (1% Safranin solution in 50% ethanol), mounted in glycerin jelly and observations were made with a Sterico research microscope under (E 40, 0.65) using a 16x eye piece, and the seeds were examined with the aid of a dissecting microscope. For SEM studies, both pollen and seeds were mounted onto stubs with double sided adhesive, and then these stubs were sputter-coated with gold (Ion-sputter JFC-1100). After coating, they were examined using JEOL JSM 5400LV scanning electron microscope at 15KV, at the Electron Microscopy Unit, Assiut University. The description of pollen follows the terminology of Hesse et al. (2009) and seed coat terminology follows Barthlott (1981). DNA barcoding Each of Dicliptera paniculata and the new species Dicliptera aegyptiaca were represented by two herbarium specimens. From each specimen, about 20mg were collected and ground under liquid nitrogen using a mortar and pestle until a fine powder was produced. DNA was extracted using a Qiagen DNeasy kit (Valencia, California, USA) as outlined by manufacturer’s protocol with few modifications for herbarium tissues. To the AP1 buffer DDT (Melford Laboratories, UK) at 0.12mg/ml and Proteinase K(Sigma) at 0.04mg/ ml were added and shaken with ground tissue for 60min at 65°C (de Vere et al., 2012). rbcl amplifications were performed following CBOL Plant Working Group (2009) with the specific primers: 5’-ATGTCACCACAAACAGAAAC3’and 5’-TCGCATGTACCTGCAGTAGC-3’ in a reaction mixture containing 25 µl PCR Master Mix (Bioline), 1µl of each primer and 20-50ng genomic DNA; the volume was completed to 50µl with sterile distilled water. The amplification protocol was 95°C for 2min followed by 34 cycles of 94°C for 1min, 55°C for 30sec and 72°C for 1min, then final extension for 7min at 72°C. Amplification products were purified using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) then sequenced with Big-dye terminator chemistry in 3130xl Genetic Analyzer (Life Technologies, California, USA) by following the standard manufacturer’s protocol. Forward and reverse sequences were assembled using Codon Code DICLIPTERA AEGYPTIACA (ACANTHACEAE), A NEW SPECIES... Aligner software, v. 7.1.2., the contig sequences were deposited in the GenBank database under accession numbers MH028051 and MG1990431.1 for Dicliptera paniculata and KU947958.1 and KU947961.1 for Dicliptera aegyptiaca. The available online sequences for Hypoestes, Peristrophe and Dicliptera in Gene bank were employed to construct a phylogenetic tree and calculate pairwise distances using Maximum Likelihood (ML) method and Cluster W in MEGA v. 6 (Tamura et al., 2013) based on Tamura 3 parameter model (Tamura, 1992) with gamma distribution. Significance was assessed using 1000 bootstrap replications (Felsenstein, 1985). Results and Discussion Dicliptera aegyptiaca E. Shamso sp. nov. Type: Egypt: Wadi El Faraied, Red Sea coast 23o31’0’’N; 35o19’60’’E; 12/2/1961; Täckholm et al. 856 (holotype CAI). Annual herb, 20– 30cm tall, unbranched, rarely with short lateral branches above. Stem angled, glabrous, sparsely strigose with appressed eglandular multicellular hairs on angles and nodes, cystolith present. Leaves rapidly deciduous at lower nodes, blackish-green when dry. Leaf lamina narrowly ovate to lanceolate 15– 45x 9– 15mm, attenuate at base, acuminate apex, minutely glandular pubescent, strigose with eglandular multicellular hairs at nerves and margins. Petiole 2– 6mm long, strigose (Fig. 1 A). Inflorescence congested in the axils with dwarf axes 1.5– 5mm long, inflorescence of (1 –) 2 – 3 cymules (inflorescence units), umbellately arranged, often compound, with many umbels in each axil. Inflorescence bracts (secondary bracts) 2, subequal, subulate, 3– 5x 0.3– 0.5mm, sparsely hairy, hyaline margin at lower half, apex acuminate. Cymule subsessile, the longest peduncle ranges from 0.5– 1mm long, subtended by 2- unequal tertiary bracts, the larger one 8– 13x 0.8– 1mm, linearlanceolate, apex sharply acuminate, the shorter 6– 9x 0.8– 1mm, linear-lanceolate, acuminate apex, pubescent with glandular and sparsely eglandular hairs and hyaline margin at lower half. Each cymule composed of 2– (– 3) flowers with 2– bracteoles each, maturing sequentially. Bracteoles as bracts, 5– 6x 0.5mm (Fig. 1 B). Calyx whitish green, sparsely hairy outside, tube 0.5– 1mm long, lobes lanceolate, 3– 4x 0.5mm attenuated to sharp acuminate apex and ciliate margin. Corolla bilabiate, resupinate 8– 8.5mm long, densely 477 eglandular hairy outside, glabrous inside, tube 3– 3.5mm long, lower lip tri-fid 4– 5x 2.5mm, upper lip retuse to acute 5– 5.5x 3mm (Fig. 1 C). Stamens 2, epipetalous, filaments 3– 5mm long, sparsely hairy, anther-thecae superposed, 0.5x 0.3mm. Pollen grains prolate, medium–sized, with mean polar axis ranges from 32.1– 35.8µm, mean equatorial diameter ranges from 20– 21.9µm, circulartriangular in polar view, heterocolpate (tricolporate with paired pseudocolpi parallel to each colporus), both colpori and pseudocolpi of about equal length (30.7– 34.6µm), colpal membrane extremely fine reticulate, endoapertures raised circular; exine sculpture foveolate to micro-reticulate (Fig. 2). Ovary oblong 1.5– 2x 0.5mm, surrounded by a cupular disc at base, attenuate at apex, eglandular hairy; style filiform, 6– 7mm long, glabrous, stigma bi-fid. Capsule 4-seeded, elliptic, 6– 7x 2mm, densely retrorse eglandular multicellular hairs, with a short rostrum (sterile portion) 1.5– 2mm long. Seeds discoid, 2x 2– 2.2mm, notched at central white hilum, seed surface variously convoluted, indistinct reticulation, covered with minute papillae and tubercles. Tubercles dense around the edges of the seed, with sharp pointed hooks in one or two rows (Fig. 3). Distribution: Confined to the southern coastal plain of the Red Sea of Egypt. Specimes examined: Wadi El Faraied, Red Sea coast 23o31’0’’N; 35o19’60’’E; 12/2/1961; Täckholm et al. 856 (holotype CAI) – Gebel Hamata, Red Sea coast, 7/2/1961; Täckholm et al. 351(CAI). Habitat: Very rare in sandy and silty moist soils. Etymology: the specific epithet aegyptiaca refers to Egypt where it was first discovered. Recognition: Dicliptera aegyptiaca closely resembles D. paniculata but differs in its stem unbranched, rarely with short lateral branches above (vs basely and laterally branched), Inflorescence congested at the nodes with dwarf axes 1.5– 5mm long, composed of (1–) 2 – 3 cymules, cymule subsessile (vs inflorescence lax panicle-like, opposite at each nodes, with long axes (20–) 30– 50mm long, composed of 3– 4 cymules, cymule pedunculate). Pollen dimention 32.1– 35.8x 20– 21.9µm, faveolate to micro-reticulate sculpture (vs pollen dimention 40x 25– 30µm, reticulate sculpture). (Fig. 1 and 2). Egypt. J. Bot. 59 , No.2 (2019) 478 EMAN M. SHAMSO AND AHMED S. FOUAD Fig. 1. Dicliptera aegyptiaca E. Shamso (Täckholm et al. 856); A: Image of specimen showing habit, B: congested inflorescence and cymule, C: Rusupinate corolla. Fig. 2. SEM micrographs of pollen grains of: Dicliptera aegyptiaca; A: Whole grain, equatorial view, B: Exine sculpture. Egypt. J. Bot. 59 , No.2 (2019) DICLIPTERA AEGYPTIACA (ACANTHACEAE), A NEW SPECIES... 479 Fig. 3. SEM micrographs of seeds of Dicliptera aegyptiaca; A: Whole seed, B: Testa sculpture. clade. DNA sequences therefore appear to help recognize groups of individual specimens and are useful for species recognition. The paraphyly of Dicliptera observed in this investigation was also recognized in other Acanthaceae genera including Acanthus using ITS sequences (McDade et al., 2000 b) and Justicia using ITS as well as plastid trnL-K, trnS-G and rps16 (Deng et al., 2016). rbcl-based DNA barcoding provided additional support for Dicliptera aegyptiaca that appeared in the phylogentic tree (Fig. 4), the tree revealed considerable interspecific variation between Dicliptera aegyptiaca and D. paniculata. The new species D. aegyptiaca clearly formed one clade strongly supported with a bootstrap value of 100% while D. paniculata along with other Dicliptera spp. formed a non-sister large 8 KR736771.1 Hypoestes sp. KX783896.1 Hypoestes phyllostachya 2 KR737526.1 Hypoestes sp. 18 10 KX783894.1 Hypoestes phyllostachya KX783895.1 Hypoestes phyllostachya 95 10 64 KR736995.1 Hypoestes sp. L12593.1 Hypoestes forsskaolii KF496829.1 Hypoestes floribunda AB586153.1 Hypoestes sp. 88 79 69 AB586152.1 Hypoestes sp. 52 KX397773.1 Dicliptera sexangularis 32 KX783902.1 Dicliptera suberecta JQ933303.1 Dicliptera obanensis 20 51 AM234782.1 Dicliptera cernua KF425772.1 Dicliptera paniculata MH028051 Dicliptera paniculata 64 21 MG199043.1 Dicliptera paniculata KU947961.1 Dicliptera aegyptiaca 100 KU947958.1 Dicliptera aegyptiaca KC825342.1 Nicotiana tabacum Fig. 4. Phylogenetic tree using Maximum Likelihood method based on Tamura 3 parameter model for rbcl sequences of Hypoestes, Dicliptera and Peristrophe spp. Bootstrap values based on 1000 replications are listed as percentages at branching points. Egypt. J. Bot. 59 , No.2 (2019) 480 EMAN M. SHAMSO AND AHMED S. FOUAD Key to the Egyptian species of Dicliptera 1- Stem unbranched, rarely with short lateral branches above. Inflorescence congested at the nodes with short axes, 1.5– 5mm long. Cymule subsessile, peduncles 0.5– 1mm long, the larger cymule-bract 8– 13x 0.8 – 1mm, linear-lanceolate, apex sharply acuminate……..……....D. aegyptiaca - Stem basely and laterally branched. Inflorescence in lax panicle- like, with axes (20–) 30– 50mm long. Cymule pedunculate, peduncles 10– 30mm long, the larger cymule-bract 12– 15x 1mm, oblong -linear, apex sharply acute… ..............D. paniculata Conclusion Results of the present investigation reflect a harmony between morphological features and DNA barcoding in delimiting D. aegyptiaca as a new species. On the other hand, appearance of D. aegyptiaca as an outgroup for a large clade containing taxa of Dicliptera and Hypoestes supports validity of DNA barcoding in species identification rather than taxonomy. After the introduction of this new species to science, the number of Dicliptera species in Egypt is raised to two. Acknowledgements: The authors thank Prof Dr. Kevin Balkwill, University of the Witwatersrand, Faculty of Science, Johannesburg, South Africa, for his critical reading, valuable comments and valuable suggestions on the manuscript. Special thanks also go to prof. Dr. Hassna Hossni, Cairo University, Egypt, for revising the manuscript. 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(Received 4/10/2018; accepted 12/3 /2019) ‫ نوع جديد من مصر مدعوما ً بالصفات‬،)‫ (العائلة األكانثية‬Dicliptera aegyptiaca rbcl ‫المورفولوجية و تقنية الباركود لألحماض النووية باستخدام جين‬ ‫ أحمد فؤاد‬،(1)‫إيمان شمسو‬ ،‫ مصر‬- ‫(المعشبة – قسم النبات والميكروبيولوجي – كلية العلوم – جامعة القاهرة – الجيزة‬1) .‫والميكروبيولوجى – كلية العلوم – جامعة القاهرة – الجيزة – مصر‬ (2) ‫قسم النبات‬ (2) ‫ كنوع جديد بمنطقة ساحل البحر األحمر بمصر وقد تناول‬Dicliptera aegyptiaca ‫أسفرت الدراسة عن وجود‬ Dicliptera paniculata ‫البحث دراسة الصفات المورفولوجية والصفات التمييزية بينه و بين النوع القريب له‬ .rbcl DNA ‫مع عمل مفتاح تعريفي لهما و تم عمل باركود للنوع الجديد ألول مرة باستخدام الشفرة الوراثية للجين‬ ‫ مللي طول‬5-1,5 ‫ بنبات ذو ساق غير متفرع و نورة ذات محور متقزم‬Dicliptera aegyptiaca ‫يتميز النوع‬. ‫ أظهرت الشجرة التطورية للشفرة الوراثية لهذين النوعين‬.‫ مللي طول‬1-0,5 ‫و نويرة محدودة ذات شمراخ قصير‬ ‫ كفرع منفرد مدعوما بتكررات تأكيد‬Dicliptera aegyptiaca ‫وجود فروق بينهما وهذا اتضح من ظهور‬ ‫ وقد أظهرت الدراسة المورفولوجية للنبات و لحبوب اللقاح و البذور إنتماء النبات الجديد لجنس‬.100% ‫بقيمة‬ ‫ بينما اظهرت الشجرة التطورية النبات الجديد فى فرع آخر غير المخصص لباقى أنواع الجنس مما‬Dicliptera .‫يظهر فائدة تقنية الباركود فى تعريف األنواع أكثر من إيجاد العالقات التصنيفية بينها‬ Egypt. J. Bot. 59 , No.2 (2019)