Pl. Syst. Evol. (2006) DOI 10.1007/s00606-006-0458-1 Seed morphology in Moehringia L. and its taxonomic significance in comparative studies within the Caryophyllaceae L. Minuto1, S. Fior2, E. Roccotiello1, and G. Casazza1 1 2 DIP.TE.RIS., University of Genoa, Italy Biology Department, University of Milan, Milan, Italy Received March 7, 2006; accepted June 13, 2006 Published online: October 24, 2006 Ó Springer-Verlag 2006 Abstract. Seeds of 30 species of Moehringia and 12 representatives of the Caryophyllaceae were examined with the Scanning Electron Microscope. High diversity was found in seed coat micromorphology, and a number of novel morphological features have been observed. Comparison with other representatives of the Caryophyllaceae has provided new insight into the potential taxonomic value of discrete morphological characters. Though many characters appear informative within Moehringia, they are affected by a high level of homoplasy when considered in a wider phylogenetic context. Smooth seed is the common condition in Moehringia, except for Eastern Balkan and Iberian species that maintain a secondary ornamentation of the testa cells resembling the plesiomorphic Caryophyllaceaetype. Five types of strophioles were recognized, in accordance with previous literature. Among these, the strophiole of the Iberian species appears very distinctive and a proper strophiole could not be verified in M. glochidisperma and M. fontqueri. Key words: Caryophyllaceae, Moehringia L., seed morphology, taxonomic significance. Introduction According to a revised classification of the genus (Hind 1988), Moehringia includes 29 recognized species, with distribution restricted to the north temperate zone. Only a limited number of species extend their distribution to North America and Asia, whereas most of the species only occur in Europe. Here, three main centres of speciation have been recognized, i.e. the Balkan Peninsula, the Iberian Peninsula and the Central European Alpine system (Hind 1988). Especially the latter, has been proposed as the main centre of endemism and speciation. The morphology of the Moehringia species and their shared similarities with Arenaria have always been considered undoubted indication of the close relationships between the two genera (McNeill 1962). Recent studies on the molecular phylogeny of the Caryophyllaceae (Fior et al. 2006) strongly support the sister position of the two genera within the subfamily Alsinoideae, but also provide indication of Moehringia being paraphyletic with Arenaria. These new results await further taxonomic studies. The literature on the genus emphasizes the scarce taxonomic significance of vegetative and floral structures, while it highlights the reliability of seed characters as possible source of information (McNeill 1962, Hind 1988). The hilar appendage of the Moehringia seeds is the L. Minuto et al.: Seed morphology in Moehringia key character to distinguish it from Arenaria (Hind 1993, Dequan et al. 2001, Rabeler and Hartman 2005), and Hind (1988) suggests a number of morphological features of the seeds worth exploring for their possible taxonomic significance, for example the testa features and cell patterning. The importance of seed morphology for classification has long been recognized (Heywood 1969, Barthlott 1984), and Echlin (1968) firstly used Scanning Electron Microscopy (SEM) photographs of seeds of Arenaria, revealing the seed microsculpturing without taxonomic comments. Since then, SEM pictures were used in systematic studies on different genera such as Sagina (Crow 1979), Arenaria (Wofford 1981, Wyatt 1984), Silene (Melzheimer 1977, Aeschimann 1984, Greuter 1995, Oxelman 1995, Hong et al. 1999), Paronychia (Ocaña et al. 1997), and Minuartia (Çelebioğlu et al. 1983). Seed micromorpholgy of Moehringia was studied in detail for isolated taxa, such as M. trinervia (L.) Clairv. (Kapil et al. 1980), and the complex group composed of Spanish representatives (viz. M. gr. intricata sensu MontserratMarti 1988), where they proved their sound taxonomic value (Diaz de la Guardia et al. 1991). Gross exomorphology has been illustrated for a number of taxa included in floristic accounts (Merxmüller and Grau 1967), as well as for some individual species (Sauer 1965, Wyatt 1984). Unlike other morphological features in Moehringia, seed characters are constant in the species (Merxmüller and Grau 1967, Hind 1988), although hardly appreciable under the light microscope. For this reason, their use has thus far been limited and their taxonomic information neglected. The present study aims to survey the seed coat micromorphology of Moehringia using SEM and provide new insights into its potential taxonomic value. Materials and methods Taxa to include in the study were selected according to the revised classification of Moehringia (Hind 1988, 1993). Seeds of all species recognized in the account were observed by Scanning Electron Microscopy (SEM), with the exception of M. hypanica Grynj & Klokov for lack of suitable material. In addition, M. fontqueri Pau (Montserrat Marti 1988, 1990), omitted in Hind’s treatment (1988), as well as the newly described M. concarenae F.Fen. & F.Martini (Fernaroli and Martini 1992) were included. For comparison and better understanding of the taxonomic value of the seed coat micromorphology, eight genera of the Caryophyllaceae were also observed, with particular emphasis on the closest relatives of Moehringia according to the phylogenetic results from Fior et al. (2006). These are mainly representatives of subfamily Alsinoideae (i.e. Arenaria, Cerastium, Stellaria, Sagina and Minuartia), as well as the Caryophylloid Silene and the Paronychioid Spergula and Paronychia. Seeds were collected in the field or from herbarium specimens. Since we noted that seed exomorphology could be subject to variation during seed development, seeds were carefully taken from mature and dehiscent capsules (Table 1). Seeds ( £ 10 per taxon) from herbarium material were air-dried on SEM stubs, sputter-coated with gold, viewed with a Philips 515 SEM at an acceleration voltage of 22 Kv, and photographed by using a Nikon Coolpix 5400 digital camera. Fresh material was previously CPD dried and later coated as above. When fresh material was available, this was certainly desirable as it yielded considerably better results, especially in regard to the preservation of the strophiole cells. In fact, these would easily collapse under the high-voltage acceleration of the SEM in case of desiccated material (e.g. M. lateriflora (L.) Fenzl - Fig. 1H). Maintaining cell turgidity under the SEM certainly eases the interpretation of the structure and anatomy of the strophiole (e.g. M. trinervia - Fig. 1T). Morphological observations and measurements were undertaken particularly of the general shape and appearance of seeds, on the testa features (i.e. epidermal cell types, papillae and ridge), and on the strophiole. Terminology follows previous study on Caryophyllaceae seeds (Hind 1988; Crow 1979; Wofford 1981; Çelebioğlu et al. 1983; Wyatt 1984; Monteserrat Martı̀ 1985, 1988; Diaz de la Guardia et al. 1991; Yildiz 2002) and recent compendia (Werker 1997). M. M. M. M. M. M. M. stellarioides Coss. lateriflora (L.) Fenzl macrophylla (Hook.) Torr. umbrosa (Bunge) Fenzl trinervia (L.) Clairv. pentandra Gay diversifolia Dolliner ex W.D.J.Koch M. M. M. M. M. M. minutiflora Bornm. intricata R.Roem. ex Willk. tejedensis Huter, Porta & Rigo ex Willk. glochidisperma J.M.Monts. fontqueri Pau lebrunii Merxm. M. M. M. M. M. M. M. M. M. M. M. grisebachii Janka pichleri Huter jankae Griseb. ex Janka villosa (Wulfen) Fenzl pendula (Waldst. & Kit.) Fenzl muscosa L. intermedia (Loisel.) Panizzi markgrafii Merxm. & Gutermann papulosa Bertol. tommasinii Marches. ciliata (Scop.) Della Torre M. concarenae F. Fen. & F. Martini M. glaucovirens Bertol. Specimens Date Legit HB Monts Babos (Kaleylii), Algeria New Beach, Glacier Pt. Garfield County, Umatilla Nat.Forest, USA Labrador, Canada Monte Ceppo, Baiardo (IM), Italy Giglio - Valle della Botte Koralpa: Glashutten, stenblock vid Mullerson Ivar Markovgrad (Macedonia) Santiago de la Espada, Sierra de Segua, Spain Salto del Caballo, Sierra Tejeda, Spain Jbel Kelti (West Rif), Morocco El Buitre-Abrucena, Sierra Nevada, Spain Vallon du Refrei, Pont De La Pia, Tende, France Dobregia, Dist. Tulcea, Locus classicus Felesenspalten im Rhodope, Bulgarien Bulgaria. Dobrudska Danubii pr. Horsava Poresen bei Podbrda, Carnia Thermos Herculis, Hungaria merid. Sciarborasca, Genova, Italy Clue de Carejuan, Gorges du Verdon, France Barghe, Val Sabbia (BS), Italy Gola del Furlo, (PU), Italy Trieste, Ospo, Italy Valle Toggia in Val Formazza, Laghi Boden, Italy Colle della Guaita, Mas. della Presolana (BG), Italy Rifugio Re Alberto, Val di Fassa (TN), Italy May 1897 13/8/1925 28/6/1997 1967 27/4/2005 29/5/1997 14/8/1959 Reverchon Anderson, ZA355 Williams, Goff 33 Neitz Minuto, Casazza Baldini Segelberg 14859/1 S S WTU WU GE FI S 5/7/1936 21/7/2005 21/7/2005 NA 24/8/1986 16/6/2004 Otto v. Ernest Rehr Minuto, Casazza Minuto, Casazza Ibn Tattou Suan 49860 Minuto, Casazza FI GE GE RAB GDA GE August 1929 28/6/1890 11/7/1909 14/8/1888 2/6/1887 18/5/2004 12/5/2004 22/6/2004 9/10/2003 July 1879 5/8/1970 Nyarady Pichler Stiefelhagen Rjavich de Degen 2316 Minuto, Casazza Minuto Minuto, Fior Casazza Marchesetti Filipello, Gentile S G W GE S GE GE GE GE S S 01/8/2005 Fior S 10/7/2003 Minuto GE L. Minuto et al.: Seed morphology in Moehringia Table 1. Taxa used in this study and voucher information. Voucher specimens are located in the following herbaria: FI Museo di Storia Naturale dell’Università, Firenze, Italy; G Conservatoire et Jardin botaniques de la Ville de Genève, Genève, Switzerland; GDA Universidad de Granada, Granada, Spain; GE Università di Genova, Genova, Italy; RAB Département de Botanique et d’Ecologie Végétale, Institut Scientifique, Rabat, Morocco; S Swedish Museum of Natural History, Stockholm, Sweden; WTU Burke Museum of Natural History and Culture, Washington DC, USA; WU Herbarium Institut für Botanik, Universität Wien, Wien, Austria; W Naturhistorisches Museum Wien, Wien, Austria Table 1. (Continued) M. bavarica (L.) Gren. M. insubrica Degen M. dielsiana Mattf. M. sedoides (Pers.) Cumino ex Loisel. Arenaria lanuginosa (Michx.) Rohrb. A. serpens Hunb., Bonpl. & Kunth Date Legit HB Pettav, Austria Between Vello and Toline, Lago di Iseo (BS), Italy Presolana, Italy Realdo (IM), Italy St. Andrew, New Castle, Jamaica Santa Cruz, Dept. Lago Argentino, Argentina 24268 Granarolo (GE) Italy Val Formazza, Italy Faeroer Islands Celle Ligure (SV), Italy Orto Botanico, Genova, Italy M. Redentore (IM), Italy M. Grai (IM), Italy Spotorno (SV), Italy Manarola (SP), Italy Isla de Majorca, Spain 16/8/1959 22/6/2004 Segelberg 16859/11 Minuto, Fior S GE 24/7/1965 01/10/2004 09/6/1959 10/1/1939 Rasetti Minuto, Casazza Webster, Ellis, Miller 8061 Eyerdam, Beetle, Grondona Minuto Gentile, Filipello Segelberg 28857/2 Minuto Minuto Minuto, Casazza Minuto, Casazza Minuto Minuto Bicknell FI GE S S 06/4/2004 05/8/1970 28/8/1952 07/4/2003 17/3/2004 21/7/2004 22/7/2004 13/7/2000 08/4/2004 21/4/1897 GE GE S GE GE GE GE GE GE GE L. Minuto et al.: Seed morphology in Moehringia A. serpyllifolia L. A. biflora L. Cerastium fontanum Baumg. Stellaria media (L.) Vill. Sagina procumbens L. Minuartia rupestris Schinz & Thell. M. verna (L.) Hiern Silene gallica L. Spergula arvensis L. Paronychia argentea Lam. Specimens L. Minuto et al.: Seed morphology in Moehringia Results Seeds of Moehringia appeared to be stable in terms of exomorphology, as only minimal intra-specific variation was found among the seeds examined for each taxon. In isolated cases where relevant variation was found, this will be specified in the text. Morphological features of the seeds such as colour, shape, testa cells and strophiole are very variable within Moehringia, and a set of relevant characters that were found distinctive for the species is reported in Table 2. The same set of characters is treated separately in Table 3 for the other Caryophyllaceae taxa examined. In the following, we will refer to the Eastern Balkan group as to include M. grisebachii Janka, M. jankae Griseb. ex Janka and M. pichleri Huter; and to the Iberian group as to include M. intricata R.Roem. ex Willk., M. tejedensis Huter, Porta & Rigo ex Willk., M. glochidisperma J.M.Monts. and M. fontqueri. Seed colour. Most Moehringia seeds show a black and shiny surface (B/s in Table 2). Exceptions were found for the Eastern Balkan (e.g. M. grisebachii) and Iberian species (e.g. M. intricata). Both groups lack brightness, but the latter also show a distinctive grey colour (G/d in Table 2). This condition was commonly found in all other Caryophyllaceae taxa observed, with the exception of the representatives of Arenaria subg. Leiosperma (e.g. A. lanuginosa (Michx.) Rohrb.) and Paronychia argentea Lam. where the shiny condition was recovered. Seed shape. In general, seed shape within Moehringia can be referred to as the crassuloid type. Lateral faces are convex, although sometimes flattened by the packing of the seeds in the capsule. A dorsal ridge may be present near the hilar zone or all around the dorsal margin. Seed shape is described (Table 2) as subcircular (s), i.e. with a central hilum and a superficial or deep notch (e.g. M. trinervia – Fig. 1Q), or reniform (r), i.e. with a subterminal hilum and a consistent superficial notch (e.g. M. muscosa L. – Fig. 4A). Other Caryophyllaceae taxa show a typical crassuloid shape (cr in Table 3) with a flat dorsal margin with papillae of variable length (e.g. Arenaria biflora L. – Fig. 6M-O, Minuartia rupestris Schinz & Thell. – Fig. 7E-G) or in some case a groove (e.g. Sagina procumbens L. – Fig. 7C). Seed coat cells. The exomorphology of the seed coat of Moehringia shows three distinctive cell patterns as a combination of primary (i.e. cellular shape and size) and secondary sculpturing (i.e. fine relief of the outer cell wall: Barthlott 1984). The most common type (22 taxa, muscosa type, see Table 2) is a cell of elongate or rounded shape lacking secondary sculpturing, showing 4–5 teeth on each side with X (e.g. M. intermedia (Loisel.) Panizzi – Fig. 4F) or in some case S-shaped contours (e.g. M. dielsiana Mattf. – Fig. 5R) resembling a jigsaw puzzle, and with an outer surface formed by flat (foveolate or alveolate) epidermal walls. This cell pattern was also recorded in Arenaria subg. Leiosperma (i.e. A. lanuginosa - Fig. 6B; and A. serpens Hunb., Bonpl. & Kunth - Fig. 6F). A second type of pattern (3 taxa, grisebachii type, see Table 2) is proper of the Eastern Balkan species, and it is formed by round cells having a weak secondary sculpturing, showing 3–4 teeth on each side with irregular contours resembling a cogwheel, and with an outer surface formed by convex (mamillate) epidermal periclinal walls (e.g. M. grisebachii – Fig. 3F). The third pattern (4 taxa, intricata type, see Table 2) was found in the Iberian species, and it is formed by cells elongate in shape and having a rough secondary sculpturing, showing 8–10 teeth on each side resembling the margins of a stamp, and with an outer surface with pronounced convex (papillate) epidermal walls (e.g. M. intricata – Fig. 2J). Except for Arenaria subg. Leiosperma, this cell pattern was recovered in all other Caryophyllaceae examined. Among these, the basic pattern was clearly discernable, but small variations were noticed. Cells may show a slightly rounded shape (e.g. Arenaria serpyllifolia – Fig. 6J), a more evident secondary sculpturing forming rough dots (e.g. Minuartia L. Minuto et al.: Seed morphology in Moehringia L. Minuto et al.: Seed morphology in Moehringia verna (L.) Hiern – Fig. 7F; Silene gallica L. – Fig. 7N), a variable number of teeth on each side with V-shaped (e.g. Stellaria media (L.) Vill. – Fig. 6V) or irregular contours (e.g. Spergula arvensis L. – Fig. 7R; Paronychia argentea – Fig. 7V), or an outer surface formed by convex (papillate) epidermal walls (e.g. Cerastium fontanum Baumg. – Fig. 6R). Papillae. A number of Moehringia species bear papillae (7 taxa – Table 2). Papillae originate as a protrusion of the testa cells. Small and simple papillae are found near the hilar zone (e.g. M. pentandra Gay – Fig. 1U-X) or on the whole surface (e.g. M. pendula (Waldst. & Kit.) Fenzl – Fig. 3U-X). In M. pentandra, intra-specific variation of this character was detected, as both conditions were recorded. Very small and simple papillae were recorded near the notch of M. pichleri (Fig. 3I-L). In the Iberian species, papillae appear more prominent, simple or often showing a more complex structure toward the apex (e.g. M. intricata – Fig. 2K; M. glochidisperma – Fig. 2S; M. fontqueri – Fig. 2W). They were recorded mainly along the marginal ridge (e.g. M. glochidisperma) and on the lateral faces of the seeds (e.g . M. intricata and M. tejedensis). Papillae were commonly found in the other Caryophyllaceae taxa examined (Table 3). Their appearance seems to vary extensively in the different taxonomic groups, and simple papillae are the most common condition (e.g. Minuartia sp. pl., Silene gallica). In Cerastium fontanum, papillae are collapsed and corrugated causing a particular cell ornamentation (Fig. 6R-S), while they become very elongated in Minuartia verna (Fig. 7J-K). Spergula arvensis (Fig. 7S) and Silene gallica (Fig. 7P) show papillae similar to the Iberian species of Moehringia. Dorsal ridge. Three main conditions of the dorsal ridge were recognized according to its extension from the hilum along the dorsal margin (Table 2): the ‘‘hilar’’ ridge covers the first third of the dorsal margin (e.g. M. concarenae – Fig. 5C); the ‘‘partial’’ ridge reaches the second third (e.g. M. papulosa Bertol. – Fig. 4O); the ‘‘complete’’ ridge is longer and can almost cover the whole dorsal margin (e.g. M. diversifolia Dolliner ex W.D.J.Koch – Fig. 2A-C). In most species of Moehringia, the ridge is constituted by mamillate cells that in the hilar region mark the break-up point of seed integuments at germination. In the Iberian species of Moehringia and in most of the other Caryophyllaceae the ridge is completely covered by papillae. A proper winged dorsal ridge was recorded only in Spergula arvensis (Fig. 7Q-S), but a similar arrangement of the cells creating a flat margin was also observed in M. trinervia (Fig. 1S), M. pentandra (Fig. 1W), and M. minutiflora Bornm. (Fig. 2E). Strophiole. As previously reported by Hind (1988), it is evident that the strophiole in Moehringia originates from the proliferation of the whitish cells of the funiculus during seed development. The variation in cell shape and their arrangement in the structure yield differences in the strophiole morphology, and five types of strophioles can be recognized (Hind 1988). In the ‘‘true amorphous’’ type (e.g. M. minutiflora – Fig. 2H) cells are not distinctly recognizable (3 taxa – Table 2). In the ‘‘umbraculiform’’ type, cells are arranged on a single layer and joined for most of their length, leaving the outermost cell walls free, thus to give the strophiole the characteristic lobate aspect (6 taxa – Table 2). In the ‘‘hyphal’’ type, cells are noticeably elongated and connected only at the very base, creating a conspicuous appendage of flexuous aspect (15 taxa – Table 2). The ‘‘papillate’’ strophiole is formed by numerous short cells of approximately b Fig. 1. Seed morphology in Moehringia. For each species general shape, cell surface, dorsal ridge and strophiole are shown. A-D M. stellarioides (A: bar= 0.5 mm; B-D: bar= 0.1 mm). E-H M. lateriflora (E: bar= 0.5 mm; F-H: bar= 0.1 mm). I-L M. macrophylla (I: bar= 1 mm; J: bar= 0,1 mm; K: bar= 0,5 mm; L: bar= 0,1 mm); M-P M. umbrosa (M: bar= 0.5 mm; N-P bar= 0.1 mm). Q-T M. trinervia (Q: bar= 0.5 mm; R-T: bar= 0.1 mm). U-X M. pentandra (U: bar= 0.5 mm; V-X: bar= 0.1 mm) Table 2. Morphological patterns in Moehringia seeds. Abbreviations: Colour: B/s black and shiny, DG/d dark grey and dull. Shape: s subcircular, r reniform. Testa cell: Gt grisebachii type: rounded, weak secondary sculpture, 3-4 teeth, irregular contours (cogwheel), mamillate surface; Mt muscosa type: elongate or rounded, no secondary sculpture, 4-5 teeth, S-shaped contours (jigsaw puzzle), foveolate surface; Mtalv as Mt but with alveolate epidermal walls; Mtirr as Mt but with irregular contours; MtX as Mt but with X contours; It intricate type: elongate, rough secondary sculpture, 8-10 teeth, zip-shaped contours (stamp), papillate surface; Itvsh as It but more rounded or elongated, V-shaped contours. Ridge: complete almost covering the whole margin; partial 2/3 covered; hilar 1/3 margin from the hilum covered Lenght (mm) Width (mm) Colour Shape Testa cell Papillae Ridge Strophiole Colour 1a-d 1e-h 1i-l 1m-p 1q-t 1u-x 2a-d 2e-h 2i-l 2m-p 2q-t 2u-x 3a-d 3e-h 3i-l 3m-p 3q-t 3u-x 4a-d 4e-h 4i-l 4m-p 4q-t 4u-x 5a-d 5e-h 5i-l 5m-p 5q-t 5u-x 1.30 1.10 1.40 1.30 0.90 1.10 0.80 0.60 0.90 1.10 1.10 0.90 1.50 0.80 0.90 1.10 1.30 1.40 1.30 1.30 1.50 1.30 1.30 0.90 0.90 1.00 1.40 1.40 1.60 1.20 1.00 1.00 1.10 1.00 1.00 0.80 0.60 0.40 0.70 1.00 1.00 0.70 1.10 0.70 0.70 0.80 0.80 1.00 0.90 1.00 1.10 1.00 0.90 1.00 1.00 0.90 1.00 1.00 1.10 1.00 B/s B/s B/s B/s B/s B/s B/s B/s DG/d DG/d DG/d DG/d B/s DG/d DG/d DG/d B/s B/s B/s B/s B/s B/s B/s B/s B/s B/s B/s B/s B/s B/s s s s s s s s s r r r r r s s s r r r r r r r r r r r r r r Mt Mt Mt Mt Mt Mt Mt Mtalv It It Itvsh Itvsh Mt Gt Gt Gt Mt Mtirr Mt MtX MtX MtX MtX MtX MtX MtX MtX MtX MtX MtX no no no no no simple no no complex complex simple/complex simple/complex no no simple no no simple no no no no no no no no no no no no complete hilar hilar hilar complete complete complete hilar absent absent absent absent partial hilar hilar hilar partial. hilar hilar hilar hilar hilar hilar hilar hilar partial hilar hilar partial hilar umbraculiform umbraculiform umbraculiform umbraculiform umbraculiform amorphous hyphal amorphous pap. complex pap. complex absent absent hyphal papillate papillate papillate hyphal hyphal umbraculiform hyphal hyphal hyphal hyphal hyphal hyphal hyphal hyphal hyphal hyphal hyphal white white white white white white white white brown brown white white white white white white white white white white white white white white white white white white L. Minuto et al.: Seed morphology in Moehringia Moehringia stellarioides M. lateriflora M. macrophylla M. umbrosa M. trinervia M. pentandra M. diversifolia M. minutiflora M. intricata M. tejedensis M. glochidisperma M. fontqueri M. lebrunii M. grisebachii M. pichleri M. jankae M. villosa M. pendula M. muscosa M. intermedia M. markgrafii M. papulosa M. tommasinii M. ciliata M. concarenae M. glaucovirens M. bavarica M. insubrica M. dielsiana M. sedoides Figure Fig. Lenght Width Colour (mm) (mm) Shape Testa cell Papillae Ridge no complete absent no hilar no complete absent simple along the ridge complete absent Arenaria. lanuginosa 6a-d 0.90 (subg. Leiosperma; A) 0.70 black/shiny s A. serpens 6e-h 0.90 (subg. Leiosperma; A) A. serpyllifolia 6i-l 0.50 (subg. Arenaria; A) 0.70 black/shiny r 0.30 grey/dull cr 0.40 dark grey/dull cr 0.90 0.70 grey/dull cr 6u-x 1.20 1.00 dark brown/ dull cr A. biflora (subg. Arenaria; A) 6m-p 0.50 Cerastium fontanum (A) 6q-t Stellaria media (A) elongate or rounded, no secondary sculpture, marked 4–5 teeth, X-shaped contours (jigsaw puzzle), foveolate surface as the previous taxon but with S-shaped contours rounded and elongate, rough secondary sculpture, 8–10 teeth, V-shaped contours (cogwheel or stamp), papillate surface elongate, rough secondary sculpture, 8–10 teeth, zip-shaped contours (stamp like), papillate surface as A. serpyllifolia but with 10–12 teeth rounded, minute secondary sculpture and rough dots, 10–15 teeth, irregular or V-shaped contours (cogwheel), papillate surface Strophiole amorphous simple mainly partial absent constituted by outer surface simple mainly complete absent constituted by outer surface L. Minuto et al.: Seed morphology in Moehringia Table 3. Morphological patterns in representative taxa of the Caryophyllaceae. Subgeneric and subfamilial classifications follow McNeill (1962) and Bittrich (1993), respectively: A = Alsinoideae; C = Caryophylloideae; P = Paronychioideae. Abbreviations: Shape: s subcircular; r reniform; cr crassuloid. Ridge: complete almost covering the whole margin; partial 2/3 covered; hilar 1/3 margin from the hilum covered Table 3. (Continued) Fig. Lenght Width Colour (mm) (mm) Shape Testa cell 0.35 grey/dull cr Minuartia rupestris (A) 7e-h 1.30 1.00 brown/dull cr M. verna (A) 7i-l 0.80 0.70 brown/dull cr Silene gallica (C) 7m-p 0.85 0.60 grey/dull cr Spergula arvensis (P) 7q-t 1.10 1.10 dark grey/dull s Paronychia argentea (P) 7u-x 1.18 1.10 brown/shiny cr simple constituted elongate or rounded, by outer surface weak secondary sculpture and rough dots, 5–7 teeth, featured with a V-shaped contours (jigsaw puzzle and stamp), foveolate surface elongate, rough simple along secondary sculpture, the ridge 8–10 teeth, zip-shaped contours (stamp), papillate surface as M. rupestris simple mainly constituted by outer surface rounded and elongate, simple along rough secondary the ridge sculpture, and near the hilum 10–12 teeth, marked V-shaped contours (cogwheel or stamp), papillate surface rounded, very rough complex on the secondary sculpture, surface only 5–6 teeth, irregular-shaped contours (cogwheel like), papillate surface elongate or rounded, absent no secondary sculpture, marked 4–5 teeth, X–shaped contours (jigsaw puzzle), foveolate surface Ridge Strophiole dorsal groove absent complete absent complete absent complete absent complete/ absent flat dorsal absent groove and wing L. Minuto et al.: Seed morphology in Moehringia Sagina procumbens (A) 7a-d 0.40 Papillae L. Minuto et al.: Seed morphology in Moehringia Fig. 2. Seed morphology in Moehringia. For each species general shape, cell surface, dorsal ridge and strophiole are shown. A-D M. diversifolia (A: bar= 0.5 mm; B-D bar= 0,1 mm). E-H M. minutiflora (E: bar= 0.1 mm; F: bar= 10 lm; G-H: bar= 0.1 mm). I-L M. intricata (I: bar= 0.5 mm; J-L: bar= 0.1 mm). M-P M. tejedensis (M: bar= 0.5 mm; N-P bar= 0.1 mm). Q-T M. glochidisperma (Q: bar= 1 mm; R-T: bar= 0.1 mm). U-X M. fontqueri (U: bar= 0.5 mm; V-X: bar= 0.1 mm) L. Minuto et al.: Seed morphology in Moehringia Fig. 3. Seed morphology in Moehringia. For each species general shape, cell surface, dorsal ridge and strophiole are shown. A-D M. lebrunii (A: bar= 1 mm; B: bar= 0.1 mm; C: bar= 0.5 mm; D: bar= 1 mm). E-H M. grisebachii (E: bar= 0.5 mm; F-H: bar= 0.1 mm). I-L M. pichleri (I: bar= 0.5 mm; J-L: bar= 0.1 mm). M-P M. jankae (M: bar= 0.5 mm; N-P: bar= 0.1 mm). Q-T M. villosa (Q: bar= 0.5 mm; R: bar= 10 lm; S-T: bar= 0.1 mm). U-X M. pendula (U: bar= 1 mm; V: bar= 0.1 mm; W: bar= 0.5 mm; X: bar= 1 mm) L. Minuto et al.: Seed morphology in Moehringia Fig. 4. Seed shape in Moehringia. For each species general shape, cell surface, dorsal ridge and strophiole are shown. A-D M. muscosa (A: bar= 0.5 mm; B: bar= 0.01 mm; C: bar= 0.5 mm; D: bar= 0.1 mm). E-H M. intermedia (E: bar= 1 mm; F-H: bar= 0.1 mm). I-L M. markgrafii (I: bar= 0.5 mm; J-L: bar= 0.1 mm). M-P M. papulosa (M: bar= 1 mm; N-O: bar= 0.1 mm; P: bar= 1mm). Q-T M. tommasinii (Q: bar= 1 mm; R-S: bar= 0.1 mm; T: bar= 0.5 mm). U-X M. ciliata (U: bar= 1 mm; V-X: bar= 0.1 mm) L. Minuto et al.: Seed morphology in Moehringia Fig. 5. Seed shape in Moehringia. For each species general shape, cell surface, dorsal ridge and strophiole are shown. A-D M. concarenae (A: bar= 0.5 mm; B-C: bar= 0.1 mm; D: bar= 0.5 mm). E-H M. glaucovirens (E: bar= 0.5 mm; F-H: bar= 0.1 mm). I-L M. bavarica (I: bar= 1 mm; J-L: bar= 0.1 mm). M-P M. insubrica (M: bar= 1 mm; N: bar= 10 lm; O-P: bar= 0.1 mm). Q-T M. dielsiana (Q: bar= 0.5 mm; R-T: bar= 0.1 mm). U-X M. sedoides (U: bar= 1 mm; V-W: bar= 0.1 mm; X: bar= 1 mm) L. Minuto et al.: Seed morphology in Moehringia Fig. 6. Seed shape in other Caryophyllaceae. For each species general shape, cell surface, dorsal ridge and strophiole are shown. A-D Arenaria lanuginosa (A-D: bar= 0.1 mm). E-H A. serpens (E: bar= 0.5 mm; F-H: bar= 0.1 mm). I-L A. serpyllifolia (I-L bar= 0.1 mm). M-P A. biflora (M-P: bar= 0.1 mm). Q-T Cerastium fontanum (Q-T: bar= 0.1 mm). U-X Stellaria media (U: bar= 0.5 mm; V-X: bar= 0.1 mm) L. Minuto et al.: Seed morphology in Moehringia Fig. 7. Seed shape in other Caryophyllaceae. For each species general shape, cell surface, dorsal ridge and strophiole are shown. A-D Sagina procumbens (A-D: bar= 0.1 mm). E-H Minuartia rupestris (E: bar= 1 mm; F-H: bar= 0.1 mm). I-L M. verna (I: bar= 0.5 mm; J-L: bar= 0.1 mm). M-P Silene gallica (M: bar= 0.5 mm; N-P: bar= 0.1 mm). Q-T Spergula arvensis (Q: bar= 0.5 mm; R: bar= 10 lm; S-T: bar= 0.1 mm). U-X Paronychia argentea (U: bar= 0.5 mm; V: bar= 10 lm; W: bar= 0.5 mm; X: bar= 0.1 mm) L. Minuto et al.: Seed morphology in Moehringia equal length (e.g. M. jankae - Fig. 3P), and it is proper of the three Eastern Balkan species (Table 2). A different kind of papillate strophiole belongs to the Iberian species (two taxa – Table 2), where brownish cells arise from a broad area around the hilar zone (e.g. M. tejedensis - Fig. 2P) and the strophiole has been suspected by different authors to be of different origin than the rest of the species of Moehringia (McNeill 1962, Hind 1988). We refer to this type of strophiole as the ‘‘papillate-complex’’. No proper strophiole was recovered in M. glochidisperma (Fig. 2T) and M. fontqueri (Fig. 2X). As expected, the other Caryophyllaceae taxa observed do not possess a strophiole, but a white remnant of the funiculus (similar to the amorphous type) was recorded in a number of seeds of Arenaria subg. Leiosperma (Fig. 6H). Discussion The use of the Scanning Electron Microscopy (SEM) in the study of Moehringia seeds has revealed a great variation in seed coat micromorphology and allowed the description of a number of novel morphological features. For these, comparison with seeds of closely related taxa within the Caryophyllaceae has provided new insight into their potential taxonomic value. Though many morphological characters would seem to be remarkably informative within Moehringia, they are affected by high level of homoplasy when considered in a wider phylogenetic context. This is not surprising considering the pronounced homoplasy that affects the Caryophyllaceae at all taxonomic levels, where only few potential synapomorphies can be pointed out (Fior et al. 2006). Seed dimensions show small variation within Moehringia, with most taxa having similar seed size. There seems to be a tendency of the narrow endemics to adopt extreme values (e.g. M. lebrunii Merxm.), whereas widely distributed species generally fall within average values. In Moehringia, no standardised measures can be pointed out to identify species, unlike other genera such as Arenaria (Wyatt 1984) and Minuartia (Çelebioğlu et al. 1983). Seed morphology of Moehringia shows some noteworthy features within the Caryophyllaceae. One of the most striking aspects is the black and shiny appearance of most of the seeds as a result of the reduced secondary sculpturing of the testa cells, which gives the seeds a distinctive smooth appearance. Exceptions are the Eastern Balkan and Iberian species, where testa cells maintain a secondary structure as is the common condition in the Caryophyllaceae (Crow 1979, Wofford 1981, Çelebioğlu et al. 1983, Wyatt 1984, Aeschimann 1984, Hong et al. 1999). However, smooth seeds are not a unique feature of Moehringia, as they were also recovered in Paronychia argentea and Arenaria subg. Leiosperma. In particular, this latter resembles Moehringia in many features, namely the shape and colour of the seeds, as well as the testa cell pattern and flatness of the outer epidermal periclinal walls. The similarities between Leiosperma and Moehringia were already noted by McNeill (1962), who also suggested a possible common ancestry of the two groups. Recent molecular results (Fior et al. 2006) show the two groups in a grade including Arenaria subg. Arenaria, and, although awaits further evidence, we suggest that McNeill’s hypothesis should not be ruled out. As mentioned before, testa cells differ in the Eastern Balkan and Iberian species, where the discernible secondary structure is combined with distinctive cell shapes. These show evident similarities with the other Caryophyllaceae representatives examined, whose seed secondary microsculpturing reveals V-shaped or irregular cell contours. Plain similarities are found with the closest relatives (Fior et al. 2006) such as Arenaria subg. Arenaria (Wyatt 1984), Stellaria and Cerastium. One of the most striking features within Moehringia is the presence of complex and conspicuous papillae in the Iberian species, which are a clear reminder of the common condition of the seeds in the Caryophyllaceae (Çelebioğlu et al. 1983) and occasionally also L. Minuto et al.: Seed morphology in Moehringia within the order Caryophyllales (Hassan et al. 2005). These structures represent a unusual feature within Moehringia, and could possibly be regarded as a plesiomorphic condition. Diaz de la Guardia et al. (1991) discussed the morphological variation of seed coat papillae in terms of ecological adaptations and geographical distribution, and supported the taxonomical value of the structures in the Iberian species. The less prominent papillae observed in this study in M. pendula, M. pentandra and M. pichleri might also be interpreted as evolutionary remnants and their variability as ecological adaptations (Hind 1988). Similarly to the secondary sculpturing of the testa, the dorsal ridge seems to have become reduced in the smooth seeds of Moehringia. In fact, while in the Iberian species the dorsal ridge is still marked by the presence of papillae as in the other Caryophyllaceae (Montserrat Marti 1988), in the rest of the species this dorsal ornamentation is characterized by mamillate cells often confined to the hilar part of the dorsal margin. At germination, this part of the seed breaks open to let the root come out. The strophiole of the seeds shows some clear variation within Moehringia, which is easily appreciable also with the use of a light microscope. The umbraculiform and hyphal types are the most common condition, whereas the papillate type characterizes the Eastern Balkan and Iberian species, though with a distinct morphology and colour in the two groups. It would seem fair to suppose that the variation of the strophiole has a reliable taxonomic value within Moehringia, but given the vital importance of the strophiole in seed dispersal, the ecological aspects should not be overlooked. In fact, there seems to be a strict correlation between the habitat in which the species grow and the type of strophiole, with the hyphal type restricted to the Alpine species growing on cliffs or overhanging rocks, and the umbraculiform to the widely distributed species and the Alpine taxa occurring on open screes. However, this does not necessarily imply a phylogenetic relationship, which should be properly tested under a cladistic framework. The two papillate forms of the strophiole which are restricted to endemic taxa of the Eastern Balkan and Iberian taxa seem to be of greater taxonomic importance. In both cases, this adds to the numerous other characters unique for the groups. As mentioned, the strophiole of the Iberian species is very peculiar in its general appearance, brownish colour and cell shape, which led McNeill (1962) to doubt on its nature and confine these taxa in a new section, i.e. Pseudomoehringia. This seems to find support in recent molecular analyses, where the Spanish representative M. intricata appears more closely related to Arenaria (Fior et al. 2006). In his treatment on the genus, Hind (1988) investigated the nature of the strophiole of the Iberian species and maintained this to be homologous to the rest of the species but with a more complex origin. We believe that the evidence here provided of the plesiomorphic exomorphology of the Iberian seeds and the anomalous strophiole should be regarded as important evidence when considering the evolutionary position of the group. In conclusion, the morphological variation of the seeds in Moehringia seems to reflect the geographical distribution of the taxa, but ecological adaptation may have played an important evolutionary role. Overall similarities in the exomorphology of the seeds point towards the recognition of four geographical and ecological groups within Moehringia, with the Alpine, Eastern Balkan and Iberian species showing the most distinct morphological adaptations compared with the widely distributed taxa. The authors thank P. O. Karis for valuable scientific advice. We wish to thank A. Corallo for the fine SEM work. Two anonymous reviewers are acknowledged for improvements on the manuscript. Research was supported by FIRBRNBE01SF project, MIUR, Italy. 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Wofford B. E. (1981) External seed morphology of Arenaria (Caryophyllaceae) of the southeastern United States. Syst. Bot. 6: 126–135. Wyatt R. (1984) Intraspecific variation in seed morphology of Arenaria uniflora (Caryophyllaceae). Syst. Bot. 9: 423–431. Yildiz K. (2002) Seed morphology of Caryophyllaceae species from Turkey (North Anatolia). Pakistan J. Bot. 34: 161–171. Addresses of the authors: L. Minuto (correspondence; e-mail: minuto@dipteris.unige.it), E. Roccotiello, G. Casazza, DIP.TE.RIS., University of Genoa, Corso Dogali 1M, 16136, Genoa, Italy. S. Fior, Biology Department, University of Milan, Via Celoria 26, 20132, Milan, Italy.