Morphological Diversity of Complex Thalloid Liverwort Genera of Sri Lanka

As part of a study initiated to trace the evolutionary relationships and biogeographic affinities of Sri Lankan complex thalloid liverworts, we here present the morphological diversity of Sri Lankan complex thalloid liverworts. This serves as the first detailed taxonomic study of the group in Sri Lanka. Specimens were collected from different geographical regions covering almost all topographic and climatic zones of the country. This study revealed 9 genera of complex thalloid liverworts in Sri Lanka. Based on our collections and identifications we prepared a generic key to Sri Lankan complex thalloid liverworts. Taxonomic descriptions were prepared for all identified genera.


INTRODUCTION
The Democratic Socialist Republic of Sri Lanka, formerly known as Ceylon, is an island in the Indian Ocean with a total land area of 65, 610 km 2 (Myers, 1990;Meegaskumbura et al., 2002;Daskon and Binns, 2010). This relatively small island claimed by Sir Arthur C. Clark as the "Island jewel of Indian Ocean" lies near the southwestern tip of India and shares the same continental shelf (Weerasooriya, 1988) (Fig. 1A).
Sri Lanka is a tropical country with a diverse topography and climate. Three major climatic zones are identified based on the mean annual precipitation; wet (2500-5500 mm of rain per annum), intermediate (1900-2500 mm) and dry (<1900 mm) (Dissanayake, 1991;Zubair, 2002) (Fig. 1C). The major topographical zones distinguished by the elevation include the central highlands (1060-2420 mm), the plains (270-1060 m) and the coastal belt (0 -270 m) (Vithanage, 1970) (Fig. 1B). There is a wide range of ecosystems due to this topographic and climatic heterogeneity of the country and the main types of ecosystems of Sri Lanka include forests, grasslands, aquatic, coastal and marine. These different ecosystems harbor a great variety of genera and species of plants and animals. The Western Ghats of India and Sri Lanka together form a biogeographic unit sharing a high endemism in flora and fauna and is one of the major biodiversity hotspots in the world (Myers, 1990;Meegaskumbura et al., 2002;Bossuyt et al., 2004). Thirty percent of Sri Lankan flowering plants are endemic to the country and nearly 75% of these occur in the Sinharaja Forest Reserve, a world heritage site designated by UNESCO (Myers, 1990). According to floristic endemism records, ~ 70% of evergreen trees, 40% of lianas and 11% of mosses are endemic to the country (Gunawardene et al., 2007;O'Shea, 2003). Many studies have been carried out on Sri Lankan flowering plants (Murawski et al., 1994;Dayanandan et al., 1999;Rubasinghe et al., 2005). However, the bryophyte flora of Sri Lanka remains poorly studied. The first checklists of bryophytes of Sri Lanka were published by B. A. Abeywickrama in 1978 as compilations of a limited number of literature Pethiyagoda, 2011;Long and Rubasinghe, 2014;Rubasinghe and Long, 2014). Historical records on Sri Lankan bryophyte collections date back to 18 th century Long and Rubasinghe, 2014;Ruklani and Rubasinghe, 2013).
Almost all the bryophyte collectors in the past were from foreign countries, and a detailed description of these collectors and their studies is given in Rubasinghe and Long (2014). A considerable percentage of bryophytes collected in the past remain in different herbaria throughout the continents and the National Herbarium of Sri Lanka contains only a few specimens (Table 1).
Although many taxonomic refinements have occurred within the past few years based on molecular phylogenetics, most of the specimens in the National Herbarium still bear older synonyms and some remain erroneously identified. There is a general scarcity of literature sources with detailed descriptions on bryophytes in Sri Lanka.  The checklist of mosses of Sri Lanka was updated in 2002 by B.J. O'Shea (O'Shea, 2002). Long and Rubasinghe updated the checklist of liverworts and hornworts of Sri Lanka in 2014. However, there are no records on endemic liverworts and hornworts and these two groups of bryophytes are the least studied groups of bryophytes in Sri Lanka (Gunawardene et al., 2007;Rubasinghe and Long, 2014).The liverwort checklist of  includes 16 species of complex thalloids in 9 genera and 4 families (Abeywickrama andJansen, 1978 a). According to the most recently updated checklist of liverworts and hornworts complex thalloids in Sri Lanka consist of 9 families; Aytoniaceae, Blasiaceae, Cyathodiaceae, Dumortieraceae, Exormothecaceae, Lunulariaceae, Marchantiaceae, Ricciaceae and Targioniaceae, 10 genera: Plagiochasma and Reboulia, Blasia, Cyathodium, Exormotheca, Lunularia,Marchantia, Riccia and Targionia and 17 species . Classification of all the liverworts in the earliest checklist  was based on Evans (1939) where Marchantia, Lunularia, Exormotheca and Dumortiera were included under the broad family Marchantiaceae and Plagiochasma and Reboulia were listed under the family Grimaldiaceae. The genus Cyathodium was included under the family Targioniaceae along with Targionia. There is no record of the genus Blasia in the checklist by , although it is mentioned in Long and Rubasinghe (2014) based on the publication of Hattori in 1968 who reported a specimen of Blasia pusilla from Sri Lanka. Complex thalloids recorded in  and Abeywickrama (1959) are based mainly on the literature "Hepaticae Indiae Orientalis" (1860) (Hepatics of Eastern India) by W. Mitten andSpecies Hepaticarum I (1898-1925) by F. Stephani. Long and Rubasinghe (2014) compiled all the available literature reports of Sri Lankan complex thalloids up to the present. However, there is no Flora for Sri Lankan bryophytes, nor taxonomic descriptions for any of the families, genera or species.
The present study was initiated to evaluate the taxonomy, phylogeny and biogeography of the islands' liverwort and hornwort flora. As a part of this pilot study we aim to present the morphological diversity of Sri Lankan complex thalloid liverworts based on new field collections.

MATERIALS AND METHODS
Taxon sampling: A thorough literature survey was carried out to trace all past collection sites of complex thalloid liverworts in Sri Lanka. Taxon sampling was made to include all past collection sites as well as new unexplored localities within the country (Fig. 2). Fresh samples were collected along with the substrate using a penknife; wrapped in paper tissues and stored in paper packets prepared according to the Schofield (1985) method. Photographs of the fresh specimens were taken in the field at the time of collection using Nikon D3200 and Nikon D3100 digital cameras. Geo-referencing data were recorded using a Garmin Global Positioning System navigator (GPS), along with locality and habitat information, collection and collectors' details. All specimens collected were initially observed using dissecting (Hertel and Reus-Optik Kassel) and light (Eruomex, Arnhem, Holland) microscopes.
Observations of detailed cellular characters were made using Olympus CX21FS1 compound microscope and Accu-scope 3025PH-BE-CS Stereomicroscope. Based on the vegetative, reproductive and spore morphological characters observed specimens were identified to their generic/specific level and a taxonomic key was prepared for Sri Lankan complex thalloid liverwort genera. Taxonomic descriptions along with photographs were prepared for all the genera identified.
Epidermal pores with several concentric rings of cells and a hyaline inner ring, or compound, rarely Taxonomic descriptions for all the specimens identified are given with relevant illustrations according to the order given in Table 2.
Lunularia is the only genus retaining under the family Lunulariaceae (Bischler-Causse et al., 2005;Long, 2006;Crandall-Stotler et al., 2009). The genus is so called because of the lunate shaped gemma cups (in Latin, lunularis= a little moon) (Bischler-Causse et al., 2005;Senning, 2006). Lunularia is a monospecific genus with the single species L. cruciata (L.) Dumort. ex Lindb. and it is recorded from Sri Lanka. Lunularia cruciata has been collected by A. H. G. Alston (1902A. H. G. Alston ( -1958 from Hakgala Botanic Gardens, Sri Lanka. Lunularia is often associated with human activities and is probably an accidental introduction from Europe. Thalli of Cyathodium are delicate in texture, shiny yellowish green or light green, not tinged with purple, the ventral surface of thalli is whitish, and plants often form incomplete rosettes (Fig. 3C, D and E). Epidermal pores are simple with 5 -7 cells in a single ring. Ventral tissue is reduced. Ventral scales are indistinct, hyaline, with or without appendages. Antheridia are embedded in the thalli. Archegonia are borne in archegonial cavities on the ventral surface near the thallus apex. Sporophyte is protected with a mostly bivalved involucre with a rim of brown pigmented cells (Fig. 3F). Spores are brown, granulate or baculate with an indistinct trilete scar.
The generic name was established in 1834 referring to cyathus which means "cup"; kyathos (ladle or cup) +-odes (similar to), i.e. similar to a cup (Bischler-Causse et al., 2005;Meagher, 2008). Srivastava and Dixit (1996) treat 11 species under the genus in which the two species, Cyathodium cavernarum Kunze and C. foetidissimum Schiffn. are pantropical (Salazar Allen and Korpelainen, 2001;Allen et al., 2004;Duckett and Ligrone, 2006) and 8 species are distributed in the Neotropics and Palaeotropics or Asia (Long, 1987;Allen and Korpelainen, 2006). There are two species recorded in Sri Lanka which are C. cavernarum and C. smaragdinum Schiffn. Ex Keissl (Srivastava and Dixit, 1996;Ruklani and Rubasinghe, 2013;Long and Rubasinghe, 2014).-Cyathodium cavernarum is not recorded by  although it is recorded by Long and Rubasinghe (2014). Srivastava and Dixit (1996) included records of C. cavernarum from a temple in Anuradhapura, Sri Lanka. There are no specimens of either two of these species in the National Herbarium (PDN).
The gametophytic thalli of Marchantia species in Sri Lanka are bluish green, yellowish green, green or dark green, sometimes tinged with purple and dichotomously branching (Fig. 4A -F). Epidermal pores compound often with 4 rings of cells with 4-8 cells. Inner pore opening round, polygonal, ellipsoid or cruciate. Ventral tissue with or without mucilage cavities. Rhizoids in 2 or 3 types. Ventral scales in 4-6 rows. Median scales with a hyaline, yellow, purplish or pink appendage with or without marginal teeth. Laminal scales with or without apical papillae. Archegoniophore stalked, receptacle symmetrical or asymmetrical, shallowly or deeply divided into terete rays or 4-10 lobes, with or without a median projection (Fig. 5A -E). Antheridiophore stalked or sessile, receptacle peltate or palmate (Fig. 5F). Involucres alternate with lobes or locate underneath lobes. Spores polymorphic or not. Gemma cups are cup-shaped with entire or ciliate lobes (Fig. 5G). Among these species only M. polymorpha and M. chenopoda remain in the genus today (Evans, 1917). There are 36 species under the genus Marchantia with a worldwide distribution (Bischler, 1998;Ho, 2013;Iamonico and Ibertite, 2013).
The genus Marchantia is subdivided into subgenera and sections according to the morphological characters of both gametophyte and sporophyte (Bischler, 1989) (Fig. 10).   refers to M. acaulis as indicated in Long and Rubasinghe (2014). Both M. amboinensis and M. palmata in Abeywickrama and Jansen (1978 a) and Ruklani and Rubasinghe (2013) are synonymized under M. emarginata (Bischler-Causse, 1989;Long and Rubasinghe, 2014). Marchantia linearis in Abeywickrama and Jansen (1978 a) is a misidentification of M. papillata subsp. grossibarba .The type specimen of Marchantia robusta was collected in Sri Lanka, however no duplicate exists in the National Herbarium, Peradeniya. According to the literature and data from the National Herbarium, Peradeniya, all records of Sri Lankan Marchantia species are collected from the Central Province and they belong to the collections of A. H. G. Alston (1902( -1958( ), M. Fleischer (1861( -1930, and F. Schmid (1811-1890). Nomenclature of most herbarium specimens has not been updated to follow the latest classifications and some of them remain as unidentified specimens. Only a limited number of specimens of the recorded species are available at the National Herbarium, Peradeniya (Table 1).  Thallus usually grayish green to green (Fig. 6A, B and C).Ventral surface of the thallus often dark purplish. Epidermal pores simple with a single ring of 4 cells.Ventral tissue without mucilage cavities. Ventral scales in two rows with ovate to lanceolate appendages. Appendages light pink to hyaline, not constricted at base. Antheridia dorsal, in cushions along the thallus mid axis (Fig. 6D).

Marchantia species recorded in Sri
Archegonia dorsal, in cushions. Archegoniophore stalk dorsal, without rhizoid furrows. Sporophyte with globose capsules turning to brown with maturity (Figs. 6E and F). Spores brownish with tuberculate areoles and a distinct trilete marking.
The name Plagiochasma is derived from combination of two Greek terms: "plagios" = lateral, and "chasma" = opening which refers to the bilabiate involucres with lateral dehiscence (Frye and Clark, 1937;Bischler-Causse et al., 2005). According to Bischler-Causse (1979) there are 30 species recognized under the genus. There are two subgenera under Plagiochasma: Plagiochasma and Micropylum (Bischler-Causse et al., 2005). Abeywickrama and Jansen (1978 a) have recorded Plagiochasma nepalense as the only recorded species under the genus. Plagiochasma nepalense (Lehm.) Steph., which was first described in Stephani (1898) is now accepted as a synonym under Plagiochasma rupestre (G.Forst.) Steph. (Bischler-Causse, 1979;Bapna and Kachroo, 2000). Plagiochasma rupestre represents the subgen. Micropylum and it is the only species recorded up to date in Sri Lanka (Bapna and Kachroo, 2000;Bischler-Causse et al., 2005;Long and Rubasinghe, 2014). The specimen of Plagiochasma collected by F. Schmid in 1954 from Mount Vernon, Central Province was determined by Sinske Hattori (1915Hattori ( -1992 a Japanese hepaticologist, as P. nepalense and a new record for Sri Lanka (Hattori, 1968). In the National herbarium of Sri Lanka all the specimens of Plagiochasma are referred to the synonymous genus Aytonia and they include the collections of A. H. G. Alston only. Aytonia rupestris G. Forst. was the basionym for P. rupestre (Stephani, 1898;Alam et al., 2013). Thalli of Reboulia green to dull green in colour, tinged with purple ( Fig. 7A -C). Epidermal cells with bulging trigones. Epidermal pores simple, with 5 to 8 cells in 4 or 5 rings, inner opening hexagonal pentagonal or spherical. Air chambers distributed thorough several layers without chlorophyllose filaments. Ventral tissue without mucilage cavities.Ventral scales in two rows, metallic purple in colour, with oil cells and 2 or 3 filiform appendages. Antheridia dorsal or terminal on main or ventral branches, in cushions (Fig. 7D). Spores brown in colour, with distinct areoles on distal face. Archegonia not observed. Fresh specimens of Reboulia have a distinct aroma and a sweet taste when crushed. The generic name is dedicated by the author to Eugen de Reboul (1781-1851), an Italian botanist from Florence (Little, 1949;Bischler-Causse et al., 2005;Meagher, 2008). The only recorded species in Sri Lanka under the genus is Reboulia hemisphaerica (L.) Raddi, the type of the genus (Bischler, 2004;Bischler-Causse et al., 2005;Long and Rubasinghe, 2014). There are herbarium specimens of Reboulia contributed by A. H. G. Alston in the National Herbarium. Alston's collections of Reboulia are recorded from Hakgala. The collection of M. Onraedt (1904Onraedt ( -1998, who was the first to record R. hemisphaerica in Sri Lanka, is not present in the National Herbarium (Onraedt, 1981). However, during the present study the species was collected from several localities.
Thalli yellowish green, bluish green or green forming rosettes or patches with or without a prominent sulcus dorsally, margin often tinged with purple both dorsally and ventrally (Fig. 7E). Epidermal pores not differentiated. Ventral tissue thick. Ventral scales purplish or hyaline in two rows. Antheridia and archegonia scattered. Sporophyte dorsally or ventrally emerging (Fig.  7F). Spores often brown in colour with or without a distinct trilete scar.

Family Sematophyllaceae Family Exormothecaceae Mu¨ ll. Frib.ex Grolle
Exormotheca Mitt., Natural History of the Azores, or Western Islands 325. 1870. Thalli of Exormotheca in Sri Lanka green or whitish green in colour (Fig. 8A, B and C). Epidermal pores are distinct and highly elevated. Air chambers contain chlorophyllose filaments. Ventral tissue with mucilage cavities. Ventral scales hyaline, without appendages. Antheridia are grouped and sunken in the median furrow of the thalli. Archegonia with 2-lobed receptacles, archegoniophore stalk with 1 rhizoid furrow ( Fig.  8D and E). Spores brown in colour, areolate with tubercules at margin.
The name Exormotheca is derived from "exormos" = extruded, "theca" = capsule, referring to the short-exserted capsules (Bischler-Causse et al., 2005).Willem Meijer (1923Meijer ( -2003 was the first person to record a species under Exormotheca in Sri Lanka. In Meijer, (1956) it is mentioned that the specimen was sent to Meijer by Prof. Abeywickrama along with some other hepatics for determination. That Exormotheca species had unique characters that deviate from the characters of other species mentioned in Schiffner's monograph of Exormotheca (Meijer, 1956). Therefore Meijer described it as a new species, Exormotheca ceylonensis which is the only recorded species under the genus in Sri Lanka (Fig.  8). Exormotheca ceylonensis is confined to the Western Ghats of India and Sri Lanka and now it is known as a threatened and rare liverwort in India (Udar and Chandra, 1964;Alam et al., 2012). Prof. Abeywickrama has collected Exormotheca from 3 localities in Sri Lanka; Kadugannawa, Hakgala and Kadadora (Meijer, 1956). Hattori, 1968 records E. ceylonensis from two other localities Galagedara and Urugala (Central Province), collected by F. Schmid.
Thalli of Dumortiera dark green in colour with bristles along the margin, dichotomously branched at the apical main thallus (Fig. 9A). Epidermal pores and air chambers absent. Ventral tissue without mucilage cavities. Ventral scales indistinct, small and hyaline, in two rows. Antheridiophore with disk shaped receptacles with bristles along the margin and dorsal side, concave at the middle, short stalked or sessile (Fig. 9B). Archegoniophores with 6-9 lobed receptacles with bristles, archegoniophore stalk with two rhizoid furrows ( Fig. 9C and D).Spores brown in colour and tuberculate proximally. The name Dumortiera was dedicated by the author to Count B. C. J. Dumortier (1797-1878), a Belgian politician and botanist for the specimens that had been previously determined as Marchantia hirsuta Sw. (Evans, 1919;Bischler-Causse et al., 2005;Gledhill, 2008). There is only one recorded species in Sri Lanka under the genus and that is Dumortiera hirsuta (Sw.) Nees. Evans (1919) mentions that some specimens from Sri Lanka seemed to be different from D. hirsuta but looked like D. irrigua (Wilson) Nees. However Stephani has included D. irrigua among the synonyms under D. hirsuta (Evans, 1919;Santarelli, 1958;Perold, 1993). Hattori (1968) records Dumortiera hirsuta var. nepalensis in the collection of F. Schmid from Kandy (Central Province), Deniyaya (Southern Province) and Pelmadulla (Sabaragamuwa Province). Checklists of Liverworts in Sri Lanka, 1978 and 2014 do not mention any varieties of Dumortiera hirsuta. But it is probable that different varieties of D. hirsuta to occur in the country.The existing herbarium specimens of Dumortiera in the National Herbarium are collections of Alston andG. Gardner (1810-1949) from Kandy district (Table 1). Thalli of Targionia is usually dull green in colour with purplish pigmentation along the margin ventrally, thalli dichotomously branched (Fig. 9E). Epidermal pores simple, with 6 to 8 cells in 2 to 3 rings. Ventral scales in two rows, dark purple and hyaline with lanceolate to ovate appendages.

CONCLUSIONS
Sir Lankan complex thalloid liverworts remained unexplored since 18 th century where A. H. G. Alston, M. Fleischer and G. Gardner have contributed much to the only remaining collections at the National Herbarium, Peradeniya to date. With the aim to initiate research on Sri Lankan complex thalloid liverworts, field excursions were made to localities of these past recorded sites as well as new unexplored localities. Some of the older locality sites (e.g. Kadugannawa, Hakgala and Nuwara Eliya) had been destroyed due to urbanization, deforestation and removal of habitats for cultivation and construction of highways.
Complex thalloids in Sri Lanka show a high morphological diversity that they differ slightly from the taxonomic descriptions of other Asiatic taxa. During the study 8 families, 9 genera and 8 species were identified. This study will be continued with molecular characterization and evaluation of biogeographic affinities of Sri Lankan liverworts. Threatened species and conservation sites will be proposed.