Systematic significance of seed morphology in Acanthophyllum (Caryophyllaceae: tribe Caryophylleae) in Iran
ATEFEH PIRANI1,*, SHAHIN ZARRE2, RICHARD RABELER3, MOSTAFA ASSADI4, MOHAMMAD REZA JOHARCHI5 & BENGT OXELMAN6
1 Department of Biology, Faculty of Science, Ferdowsi University of Mashhad, P.O. Box 91775-1436, Mashhad, Iran; email: [email protected] Department of Plant Biology, and Center of Excellence in Phylogeny, School of Biology, College of Science, University of Tehran, P.O. Box 14155-6455 Tehran, Iran3 University of Michigan Herbarium - EEB, 3600 Varsity Drive, Ann Arbor, Michigan 48108-2228, U.S.A.4 Research Institute of Forests and Rangelands, Agricultural Research Education and Extension Organization (AREEO), P. O. Box 13185-116, Tehran, Iran5 Department of Botany, Research Center for Plant Sciences, Ferdowsi University of Mashhad, P.O. Box 91775-1436, Mashhad, Iran6 Department of Biological and Environmental Sciences, University of Gothenburg, Box 461, 40530 Göteborg, Sweden* Author for correspondence
Abstract
Acanthophyllum, with ca. 90 spiny cushion-forming species, is one of the largest genera of Caryophyllaceae. Although taxonomic utility of seed morphology has already been highlighted in different genera of Caryophyllaceae, the systematic value of seed characters in Acanthophyllum has not been adequately addressed. In order to evaluate the application of seed morphology in infrageneric classification of Acanthophyllum, we surveyed seed characters in the five Iranian sections of Acanthophyllum. Seed morphology of 32 accessions of Acanthophyllum representing 21 species and four sections were investigated using scanning electron microscopy. Seed morphological characters of two species from one additional section were included based on previous studies in the Caryophyllaceae. Seeds in the majority of examined species are oblong in outline. Five types of seed surface can be considered: reticulate, reticulate-papillate, colliculate, colliculate-papillate, and colliculate-columellate. Papillae type is a reliable character for separation of certain natural groups within Acanthophyllum. Seed features provided strong evidences for separating sections, especially the following ones: Acanthophyllum, Macrostegia and Pleiosperma, whose seeds showed a high degree of uniformity among the sampled species. Morphological characteristics of seeds were less useful for discriminating Acanthophyllum species. While seed characters are consistent within individual populations, they can vary among different populations of an individual species. Our results show that variation in seed morphological characters are in agreement with phylogenetic patterns within Acanthophyllum.
Keywords: Acanthophyllum, infrageneric classification, phylogeny, scanning electron microscopy, seed-coat microsculpturing
Introduction
Acanthophyllum Meyer (1831: 210) in the broad sense (sensu Pirani et al. 2014), includes ca. 90 species with Irano-Turanian distribution pattern extending from Syria to Western China (Bittrich 1993, Ghaffari 2002, 2004). The genus is mostly diverse in northeast Iran and the neighboring areas of Afghanistan, and Turkmenistan (Ghaffari 2004). Most of Acanthophyllum species are cushion-forming subshrubs armed with spiny leaves, except for the small sections Allochrusa (Bunge ex Boiss.) Pirani & Rabeler (2017: 198), Ochotonophila (Gilli) Pirani (2014: 604), and Paniculata Golenkin (1893: 86) that possess non-spiny leaves. Based on a molecular phylogeny of Caryophyllaceae by Harbaugh et al. (2011) it was suggested a tribal classification including 11 monophyletic tribes, which was later confirmed by Greenberg & Donoghue (2011), and replaced the earlier classification system of the family (e.g., Bittrich 1993) recognizing three subfamilies Alsinoideae Fenzl, Caryophylloideae Arnott and Paronychioideae Meisner. Acanthophyllum is currently placed in tribe Caryophylleae Lamarck & Candolle (1806: 386), sister to tribe Sileneae Candolle ex Seringe (1824: 351).
Acanthophyllum has been subject of four main taxonomic treatments (see e.g., Boissier 1867, Golenkin 1893, Schischkin 1936, Schiman-Czeika 1988). Pirani et al. (2014) used molecular data to evaluate generic boundaries and infrageneric classifications of the genus. They suggested that the genus is monophyletic if it is defined in a broad sense, including a few small genera such as Allochrusa Bunge (Boissier 1867: 559), Ochotonophila Gilli (1956: 69), and Scleranthopsis Rechinger (1967: 37) which were previously treated as separate taxa. Moreover, Pirani et al. (l.c.) revealed two major clades within Acanthophyllum s.l. that are characterized by different petal morphology (entire versus emarginate petals). Accordinly, Acanthophyllum s.l. should include at least 11 sections among which sect. Acanthophyllum (ca. eight species), sect. Allochrusa (ca. six species), sect. Macrostegia (Boissier) Pax (1889: 74) (ca. 10 species), sect. Oligosperma Schischk. ex Schiman-Czeika (1988: 292) (ca. 30 species), and sect. Pleiosperma (Boissier) Pax (1889: 74) (ca. six species) occur in Iran [the name of one, and authorship of three sections differ from those given in Pirani et al. (2014); for an explanation see Pirani and Rabeler (2017) and Rabeler (1993)]. These five sections, including a total of 40 species in Iran, are recognized by differences in reproductive and vegetative features, such as the number of ovules per capsule and inflorescence characteristics. As a successful accessory approach, seed morphology has increasingly been used in recent taxonomic studies of Caryophyllaceae (e.g., Minuto et al. 2006, Minuto et al. 2011, Amini et al. 2011, Kanwal et al. 2012, Sadeghian et al. 2015, Hoseini et al. 2017, Arabi et al. 2017). These studies have provided valuable information for delimitation of taxa at generic, infrageneric and specific ranks. Notably, a large number of studies on seed-coat microsculpturing in Caryophyllaceae have focused on tribes Alsineae Candolle in Lamarck & Candolle (1815: 766), Arenarieae Rohrbach (1872: 262), Sagineae Tanfani (1891: 378), and Sileneae (see e.g., Melzheimer 1977, Crow 1979, Wofford 1981, Wyatt 1984, Çelebioğlu et al. 1983, Hong et al. 1999, Minuto et al. 2006, 2011, Mahdavi et al. 2012 Arman & Gholipour 2013, Mostafavi et al, 2013, Sadeghian et al. 2014, Dadandi & Yıldız 2015, Hoseini et al. 2017, Arabi et al. 2017). Comparative seed morphological studies in tribe Caryophylleae have been performed sporadically, among which seed morphological features of some genera could be obtained from the larger studies on the whole family, where the included genera are represented by a few species (e.g., Yıldız 2002, Kanwal et al. 2012). For example, Dianthus Linnaeus (1753: 409), Petrorhagia (Ser.) Link (1829: 235), and Saponaria Linnaeus (1753: 409) have been included with poor sampling (only one to three species) in recent seed morphological studies (Yıldız 2002, Kanwal et al. 2012), and the value of seed morphology for taxonomy of the mentioned genera has not investigated yet. Eröz Poyraz & Ataşlar (2010) reported the seed morphological characteristics of five Velezia Linnaeus (1753: 409) species in Turkey indicating their credibility for differentiating Velezia species. The most comprehensive seed morphological study in the tribe that has been carried out on Gypsophila Linnaeus (1753: 406), included 22 species from Iran (Amini et al. 2011). The latter study also included five species of Saponaria as well as two species of Acanthophyllum sect. Allochrusa (treated there under the genus Allochrusa), and suggested that seed features are useful for separation of Allochrusa from Gypsophila, and Saponaria. Bülbül et al. (2017) studied seed morphology of five Acanthophyllum species in Turkey, and provided a description of the surveyed features without discussion on taxonomic utility of seed characters. However, Acanthophyllum has not yet been the subject of a comprehensive seed morphological investigation and the systematic value of seed characters is not clear in the genus. The main reasons for paucity of seed morphological studies in Acanthophyllum, are the low seed set in these plants, occurring probably due to chromosomal failures raised through ploidy or chromosomal aberrations (Ghaffari 2004) or harsh environmental conditions of subalpine steppes that prevent successful pollination, fertilization or embryo development. Here we perform a seed micro- and macromorphological investigation on five sections of Acanthophyllum distributed in Iran which with 42 species and 12 endemics is one of the main diversification centers of the genus. The goals of this study are: 1) to document seed morphological characters in the selected sections of Acanthophyllum, 2) to investigate the application of seed morphology in systematics of examined Acanthophyllum taxa particularly at specific and sectional ranks, 3) to assess the correlation between seed morphological characters and phylogenetic patterns affecting the examined taxa of Acanthophyllum.
Material and methods
Seed samplingSeed sampling was focused on Acanthophyllum sections occurring in Iran (sectional concept follows Pirani et al. 2014). Mature seeds of available taxa from four sections (Acanthophyllum, Macrostegia, Oligosperma and Pleiosperma) were collected during field surveys to different parts of Iran or removed from herbarium specimens. Seed characters of
section Allochrusa, a fifth section, were included based on Amini et al. (2011). In order to investigate possible intra-specific variation of the seed characters in Acanthophyllum, we aimed to sample several populations of individual species. Seven species were represented with at least two populations (Table 1). To ensure the consistency of seed characters, at least 10 seeds from each population were examined. Herbarium specimens of the studied taxa (Table 1) are deposited at FUMH, TARI, TMRC, and TUH (acronyms follow Thiers 2018+).
Microscopic analysesAll seed samples were first examined by light microscopy to make sure that seeds were not damaged or wrinkled. Seed size and shape were investigated using a Zeiss Axioskop 40 light microscope (Germany). The mean value and standard deviation of the size parameters were calculated for every accession. The upper surface of testa cells was surveyed using Scanning Electron Microscopy (SEM). Selected seeds were air-dried and fixed on aluminum stubs using double-sided adhesive tape and sputter coated with gold. The SEM micrographs were taken using a LEO 440I (UK) at an accelerating voltage of 20 kV. The region used for taking close-up micrographs is indicated by a blue box in Fig. 1A.A total of nine characters (shape, length, width, color, sculpturing pattern, shape of testa cells, testa cell margin, outer periclinal walls, and depth of anticlinal walls) were investigated. Previous studies in Caryophyllaceae (e.g., Amini et al. 2011, Sadeghian et al. 2014, Hoseini et al. 2017) have shown that these characters are most informative and reliable. The terminology follows Crow (1979), Barthlott (1981), Yıldız (2002), Minuto et al. (2006), Moazzeni et al. (2007), Amini et al. (2011) and Sadeghian et al. (2014) with some modifications.
Results
The SEM micrographs here presented (Figs. 1–2) are sorted in alphabetic order of species names within each section. Details of the nine selected seed characters investigated for each taxon are also provided (Table 2). The mean of seed length varied between 1.5 and 2.5 mm, with Acanthophyllum spinosum (Desfontaines) Meyer (1831: 210) having the shortest, and A. leucostegium Schiman-Czeika (1988: 272) having the longest seeds. Regarding seed width, A. acerosum Sosnowsky (1915: 7), A. gracile Bunge ex Boissier (1867: 562), A. leucostegium, A. squarrosum Boissier (1853: 81) and A. spinosum have the narrowest seeds, with a mean about 1.0 mm, whereas A. crassinodum yukhananov & Edmondson (1977: 110) and A. adenophorum Freyn (1903: 867) possessed the broadest seeds with the mean about 1.6 mm.
Five seed-coat sculpturing patterns were recognized: 1) Reticulate (here reported as “Ret”): a net of testa cells characterized with raised anticlinal walls, testa cells lack any projections (e.g., Fig. 1M); 2) Reticulate-papillate (“Ret-pap”): a net of testa cells characterized with raised anticlinal walls, testa cells ornamented with papillae (e.g., Fig. 1H); 3) Colliculate (“Col”): periclinal walls of testa cells are flat or slightly convex, anticlinal cell boundaries are furrowed, testa cells lack projections (e.g., Fig. 2N); 4) Colliculate-papillate (“Col-pap”): periclinal walls of testa cells are flat, anticlinal cell boundaries are furrowed, testa cells ornamented with papillae (e.g., Fig. 1D); 5) Colliculate-columellate (“Col-cll”): periclinal walls of testa cells considerably raised and arranged as closely located cylindrical, dome-shaped or irregular elevations, anticlinal cell boundaries are furrowed (e.g., Fig. 1R). Seed shape, color, sculpturing pattern and testa cell features were consistent within individual populations. Intra-specific variation of the seed shape is observed in two species, A. korshinskyi Schischkin (1932: 39), and A. spinosum. The obtained results for seed characters are described for each section as below.
Section AcanthophyllumInvestigated seed features show high degree of uniformity among species of section Acanthophyllum. The examined species have dull brown seeds, oblong (e.g., Fig. 1A) or oblong-ellipsoid (e.g., Fig. 1C) in outline. Sculpturing pattern is Col-pap. Papillae are enlarged and collapsed. The surface of papillae is striate. The testa cells are elongated (e.g., Fig. 1B) and have dentate margins (e.g., Fig. 1B). The anticlinal walls of testa cells are deep (e.g., Fig. 1F). The only species of this section represented by more than one population is A. acerosum. Seed characters do not show variability among two different populations of this species (Table 2).
A. adenophorum OligospermaProv. Khorassan, Kalat-e Naderi, Beginning of Garu road, Joharchi & Zangooei 36810 (FUMH)
A. brevibracteatum Oligosperma Prov. Khorassan, NW Dargaz, Zarrinkuh protected area Amiri, s.n. (FUMH) A. caespitosum Oligosperma Prov. Kermanshah, Bisotun, Attar et al., 19961 (TUH)
A. ejtehadii OligospermaProv. Khorassan, Mashhad to Kalat-e Naderi, 125km to Kalat-e Naderi, Moazzeni & Pirani 2151 (TMRC)
A. heratense Oligosperma Prov. East Azarbaijan, Oskou, Oskou to Gonbarf village, Moazzeni & Pirani 2180 (TMRC)A. heratense Oligosperma Prov. Semnan: Damghan to Semnan, 49 km to Semnan, Moazzeni & Pirani 41070 (TUH)A. heterophyllum Oligosperma Prov. Kerman, Sirjan to Bardsir, 77–85 km to Bardsir, Moazzeni & Pirani 2921 (TMRC)A. korshinskyi Oligosperma Prov. Khorassan, 27 km to Darvarzan from Sabzevar, Moazzeni & Pirani 2178 (TMRC)
A. korshinskyi OligospermaProv. Khorassan, Mayamey to Shahrud, 2 km after Mayamey, Moazzeni & Pirani 2507 (TMRC)
A. korshinskyi OligospermaProv. Khorassan, old road of Mashhad to Neyshabur, 5 km to Molkabad, Pirani & Moazzeni 2183 (TMRC)
A. korshinskyi OligospermaProv. Khorassan, Mashhad to Neyshabur, 70 km to Neyshabur, sharifabad village,Pirani & Moazzeni 41072 (TUH)
A. laxiusculum OligospermaProv. Khorassan, Soltanabad to Sabzevar, 40 km to Sabzevar, Moazzeni & Pirani 2187 (TMRC)
A. pachystegium Oligosperma Prov. Khorassan, NE of Bojnurd, 27 km to Raz, Moazzeni & Pirani 3044 (TMRC)A. speciosum Oligosperma Prov. Khorassan, Dargaz, Chehelmir, Moazzeni & Pirani 2155 (TMRC)A. squarrosum Oligosperma Prov. Semnan, Damghan to Semnan, 49 km to Semnan, Moazzeni & Pirani 2124 (TMRC)A. crassinodum Pleiosperma Prov. Kerman, Rayen, Hezar mount., toward camping, Moazzeni & Pirani 2923 (TMRC)A. glandulosum Pleiosperma Prov. Mazandaran, Firuzkuh road, Amin-Abad village, Mehregan et al. 2508 (TMRC)A. glandulosum Pleiosperma Prov. Semnan: Semnan to Damghan, 72 km to Damghan, Moazzeni & Pirani 41706 (TUH)
A. glandulosum PleiospermaProv. Tehran. After Damavand to Firuzkuh, 1 km to Seyyed-Abad, Moazzeni & Pirani 41875 (TUH)
A. sordidum PleiospermaProv. Semnan, Damghan to Cheshmeh-Ali, Astaney-e Cheshmeh-Ali village, Pirani & Moazzeni 41709 (TUH)
A. spinosum PleiospermaProv. Isfahan, Saman to Rezvanshahr, 7 km to Rezvanshahr, Moazzeni & Pirani 2150 (TMRC)
A. spinosum Pleiosperma Prov. Fars, Neyriz to Sirjan road, 41 km to Gatrooyeh, Moazzeni & Piarni 2894 (TMRC)A. spinosum Pleiosperma Prov. yazd, Mehriz, Darre Damgahan, Mozaffarian 77824 (TARI)
FIGURE 1. Scanning electron micrographs of seeds. A, B. A. acerosum (sect. Acanthophyllum); C, D. A. crassifolium (sect. Acanthophyllum); E, F. A. microcephalum (sect. Acanthophyllum); G, H. A. bracteatum (sect. Macrostegia); I, J. A. gracile (sect. Macrostegia); K, L. A. leucostegium (sect. Macrostegia); M, N. A. adenophorum (sect. Oligosperma); O, P. A. brevibracteatum (sect. Oligosperma); Q, R. A. caespitosum (sect. Oligosperma); S, T. A. ejtehadii (sect. Oligosperma).
Section MacrostegiaSeed characters are uniform among species of section Macrostegia. The investigated seeds are dull brown, and oblong in shape. Sculpturing patterns is Ret-pap. The surface of papillae is striate. The testa cells have irregular (e.g., Fig. 1J) shapes and puzzle-shaped margins (e.g., Fig. 1H). The anticlinal walls of testa cells are indistinct (e.g., Fig. 1J). Two species in section Macrostegia, A. bracteatum Boissier (1843: 43) and A. leucostegium, were represented by two populations. Inter-population variability of seed features is not observed in these taxa (Table 2).
Section OligospermaSeed characters show low degree of uniformity among species of section Oligosperma. Observed seeds are dull brown and oblong, oblong-ellipsoid, or oblong-ovoid (e.g., Fig. 2C) in outline. Sculpturing patterns are Ret, Col and Col-cll,
among which the Ret type is the most common. Testa cells are irregular in shape and elongated. Testa margins can be dentate, puzzle-shaped, undulate (e.g., Fig. 2H), and Ω-undulated (e.g., Fig. 2L). The anticlinal walls of testa cells are indistinct, with exception of A. caespitosum Boissier (1843: 42) and A. laxiusculum Schiman-Czeika (1988: 299) which are characterized by deep anticlinal walls. Acanthophyllum korshinskyi and A. heratense Schiman-Czeika (1988: 304) are represented by more than one population in this study. Seed shape is variable among four different populations of A. korshinskyi, and this species is presented by dimorphic seed shape (Table 2). Three of the examined populations of this species possessed oblong-ovoid seeds (not shown), and one showed oblong seeds (Fig. 2F). Intra-specific variation in seed characters was not observed based on two examined populations of A. heratense (Table 2).
FIGURE 2. Scanning electron micrographs of seeds. A, B. A. heratense (sect. Oligosperma); C, D. A. heterophyllum (sect. Oligosperma); E, F. A. korshinskyi (sect. Oligosperma); G, H. A. laxiusculum (sect. Oligosperma); I, J. A. pachystegium (sect. Oligosperma); K, L. A. speciosum (sect. Oligosperma); M, N. A. crassinodum (sect. Pleiosperma); O, P. A. glandulosum (sect. Pleiosperma); Figs. Q, R. A. sordidum (sect. Pleiosperma); S, T. A. spinosum (sect. Pleiosperma).
Section PleiospermaSeed features show relatively high degree of uniformity among species of section Pleiosperma. The seeds in examined taxa of section Pleiosperma have dull brown, shiny black, and shiny brown colors. Seeds are reniform (e.g., Fig. 2M), reniform-ovoid (e.g., Fig. 2O), and oblong-ovoid in outline. Sculpturing patterns is Col (in most species) and Ret (only in A. sordidum Bunge ex Boiss. (1867: 565)). Testa cells are irregularly elongated (with exception of A. sordidum bearing non-elongated testa cells). Testa cell margins are dentate (in most species) and Ω-undulated (in A. sordidum). The anticlinal walls of testa cells are shallow (e.g., Fig. 2P) or indistinct. Two species in section Pleiosperma, i.e., A. spinosum and A. glandulosum Bunge ex Boissier (1867: 565), were represented by three populations. Seed shape is variable among different populations of A. spinosum which show dimorphic seed shape. Two of the examined populations of A. spinosum showed oblong-ovoid seeds (Fig. 2T), while one has reniform-ovoid (not shown) seeds. Investigated characters of seeds do not show variability among three different populations of A. glandulosum (Table 2).
Discussion
Only a limited number of Acanthophyllum taxa have been included in previous seed morphological studies. Bülbül et al. (2017) investigated seed morphology of five species of section Acanthophyllum in Turkey, following a descriptive style, and did not perform any comprehensive attempt on other related sections, nor explicitly defined the diagnostic characters of the studied species. However, our morphological observations for Iranian Acanthophyllum taxa are in agreement with observations of Bülbül et al. (2017) for Turkish taxa. Amini et al. (2011) reported that seed coat papillae exist in section Allochrusa. They described seed shape as helicoid, and testa margin as straight in this section. However, based on the figures presented by Amini et al. (l.c.), we suggest that outline of seeds in this group varies from reniform to subcircular, testa cell margins are dentate, and seed coat pattern is Col and Col-pap. Based on the previous studies, seed shape in Acanthophyllum documented as reniform to obovoid (Bittrich 1993). The present work shows that oblong seed shape is one of the common outline types within the genus, but it has not been reported in relatives of Acanthophyllum such as members of the genera Gypsophila, Saponaria and Dianthus. Therefore, seed shape could limitedly be used for discriminating Acanthophyllum from its related genera.
Systematic implication of seed charactersSections Acanthophyllum, Macrostegia, and Oligosperma have oblong and semi-oblong seeds. Section Pleiosperma is characterized by reniform to semi-reniform seeds, and section Allochrusa is marked by reniform to subcircular seeds. Therefore, seed shape (reniform to semi-reniform) could be used as useful character to separate taxa of sections Allochrusa and Pleiosperma from other groups of the genus. The majority of taxa studied here possess dull brown seeds. Shiny brown, and shiny black seeds were observed in three taxa of section Pleiosperma, i.e., Acanthophyllum crassinodum, A. glandulosum, and A. sordidum. Interestingly, all of these three taxa are among the late-flowering species that flower two to four weeks later than the other species of the genus in the same geographical area (A. Pirani pers. observ.). The shiny seed condition is helpful in recognizing members of section Pleiosperma. Sculpturing pattern is also consistent within examined sections, with two exceptional cases in section Oligosperma. All studied taxa in this section have Ret type sculpture, except for Acanthophyllum caespitosum (Col-cll type; Fig. 1R) and A. laxiusculum (Col type; Fig. 2H). Moreover, A. caespitosum is the only species of section Oligosperma that has emarginate petals. Procumbent densely branched habit, extremely short stems and imbricate leaves are additional characters present only in A. caespitosum. Inclusion of A. caespitosum in section Oligosperma has also been debated by previous studies (Ghaffari 2004, Pirani et al. 2014). The present data provide additional evidence for excluding A. caespitosum from section Oligosperma. Although shape of testa cells varies among sections, this variation is not informative for separation of taxa at sectional level. Variation of this character can limitedly be used to distinguish certain species within sections. For example, elongated testa cells differentiate A. laxiusculum, and A. heratense from the rest of section Oligosperma. Testa cell margins are highly uniform within sections Acanthophyllum, Macrostegia, and Pleiosperma, while considerably variable in section Oligosperma. Section Oligosperma is the largest and taxonomically most diverse group within the genus. The results obtained by us indicate that morphological diversity in section Oligosperma is well reflected by considerable variation in testa cell margin. The testa cell margin is limitedly helpful at specific level,
and provides useful information for separating A. laxiusculum, and A. heratense, two closely related species with overlapping borders in section Oligosperma. Morphologically, these taxa are highly similar. Flowers in A. laxiusculum are pedicellate, and partial cymes are pedunculate, while A. heratense has sessile flowers, and partial cymes are sessile. These characters could not easily be recognized in different populations of these taxa. Hence, some authors consider these species to be synonym. For example, in the taxonomic revision of Acanthophyllum in Iran, Basiri-Esfahani et al. (2011) reduced A. heratense under synonymy of A. laxiusculum. However, our results provide supportive evidence for treating these species as separate.
FIGURE 3. Key seed features and morphological synapomorphies of the studied Acanthophyllum sections are summarized on a simplified phylogenetic tree of Acanthophyllum s.l. adopted from Pirani et al. (2014). Sectional placement follows Pirani et al. (2014). Selected characters are mentioned below the nodes. Seed characteristics of section Allochrusa are based on Amini et al. (2011). Colored boxes highlight the sections discussed in this study. Species in quotes indicate taxa that their exclusion from the currently assigned sections is supported by seed characters. * indicates parallel evolution (convergence); ** indicates reversal.
Two different types of seed coat papillae (see above under results) were observed in the present study. According to Amini et al. (2011), a third type of papillae can be recognized within section Allochrusa. Unlike the papillae observed in sections Acanthophyllum, and Macrostegia, papillae in sect. Allochrusa are not collapsed and emerge as a rounded projection with flat surface, from the middle part of the elongated testa cells. Variation of papillae has been recorded in other genera of the Caryophyllaceae such as Arenaria Linnaeus (1753: 423), Gypsophila, Moehringia Linnaeus (1753: 309), and Paronychia Miller (1754: without page) Wofford 1981, Diaz de la Guardia et al. 1991, Minuto et al. 2006, Amini et al. 2011, Sadeghian et al. 2014). Diaz de la Guardia et al. (1991), and Minuto et al. (2006) discussed that morphological variation of seed coat papillae in Moehringia could be interpreted as reflective of geographical distribution and ecological adaptations of different species. However, our results for Acanthophyllum indicate that geographic or ecological factors might not have been effective in evolution of papillae within the genus. Instead, it seems that variation of papillae in Acanthophyllum s.l. is reflective of phylogenetic signals, and papillae type is a reliable tool for differentiating certain natural groups within the genus.
Apart from Acanthophyllum caespitosum, periclinal walls of testa cells are flat in all studied taxa. Anticlinal walls of testa cells in majority of the species are indistinct. Deep anticlinal walls help to discriminate species of section Acanthophyllum as well as A. laxiusculum and A. caespitosum (both belonging to section Oligosperma) from the rest of studied species. Shallow anticlinal walls mark examined species of section Pleiosperma except for A. sordidum. In section Pleiosperma, Acanthophyllum sordidum is characterized by different petal morphology which are non-clawed linear emarginate, while the other species of the section have clawed entire petals. This species is reported to be tetraploid, while the other examined species of the section are hexaploid (Ghaffari 2004). Exclusion of A. sordidum from section Pleiosperma was suggested also by molecular phylogeny (Pirani et al. 2014), and is supported here by its differentiating seed features (i.e., testa cell shape and margin, and anticlinal walls of testa cells). Variation in seed size was not useful for infrageneric classification of the examined Acanthophyllum taxa. Our study on seeds of Acanthophyllum showed that characters such as shape and color as well as features of testa cells, at least for the sections examined, provide a set of reliable characters to delimit traditional sections. However, variation of seed characters in other Caryophyllaceous genera such as Arenaria (Sadeghian et al. 2014), Cerastium Linnaeus (1753: 437) (Arabi et al. 2017), Gypsophila (Amini et al. 2011), and Silene Linnaeus (1753: 416) (Dadandi & Yıldız 2015) do not provide informative data for delimiting traditional sections defined by classical taxonomy.
Correlation between phylogenetic patterns and seed coat characters Variation of seed morphological characters in studied sections of Acanthophyllum agrees well with the phylogenetic patterns reflected by molecular investigation of the genus (see Pirani et al. 2014). The diagram of key morphological features as well as seed characteristics in five Acanthophyllum sections is summarized on the simplified phylogenetic tree of the group obtained from Pirani et al. (2014) (Fig. 3). Seed characteristics of section Allochrusa on the Fig. 3 are listed based on Amini et al. (2011).
CLADE IClade I includes taxa with entire petals. Three of the investigated sections, i.e., Pleiosperma, Macrostegia, and Oligosperma are nested in Clade I (Fig. 3; subclades A, B and C, respectively). Both papillate and non-papillate seeds can be found in Clade I. Seed characteristics of these sections are highly consistent with their placement in the corresponding subclades. Reniform (to reniform-ovoid) and shiny seeds are demonstrative features of subclade A that embraces section Pleiosperma. Representatives of this section possess the highest ovule number (≥ 8) and highest ploidy level (hexaploid) reported in the genus (Ghaffari 2004, Pirani et al. 2014). It appears that the general trend of seed evolution in section Pleiosperma is the shift from a dull to shiny seed surface as well as development of reniform (to reniform-ovoid) seeds. Papillate seed surfaces, four ovules per ovary and membranous bracts/bracteoles characterize subclade B containing the members of section Macrostegia. The key seed features in section Macrostegia are oblong seeds, puzzle-shaped testa margins and collapsed papillae. Subclade C holds representatives of section Oligosperma whose members possess compound cymes, pink to lilac petals and 4 ovules per ovary. The section includes several morphologically similar species with blurred interspecific borders. Analyses of nuclear ribosomal internal transcribed spacer (ITS) and the chloroplast gene rps16 sequence data (Pirani et al. 2014) did not resolve phylogenetic relationships among species of this section. Similarly, seed character variation is of only limited use in species delimitation. So, the evolutionary trends within this section remain obscure.
CLADE IIThe taxa resolved in Clade II have emarginate petals. The main components of the Clade II are sections Allochrusa, and Acanthophyllum, corresponding to subclades D and E, respectively. Acanthophyllum caespitosum (section Oligosperma) and A. sordidum (section Pleiosperma) are nested within the Clade II as well. Existing of papillae on seed surface is one of the major differentiating seed characters in Clade II. With the exception of A. sordidum, and A. caespitosum, papillae are present in the other components of Clade II, suggesting at least two reversals for this character in A. sordidum and A. caespitosum. Subclade D includes representatives of section Allochrusa. Enclosed stamens and non-spiny leaves discriminate this section from the other sections in Iran. Evolution of seed features in this section has led to development of reniform to subcircular seeds ornamented with non-collapsed papillae (Amini et al. 2011). As mentioned above, reniform seeds are also found in Clade I (section Pleiosperma). This suggests that reniform seeds have arisen at least twice within Acanthophyllum s.l.
Subclade E holds taxa of section Acanthophyllum. Species of this section could be recognized by branched elongated inflorescences with white flowers. Evolutionary trends of seed characters in this subclade are mainly: elongated testa cells possessing enlarged collapsed papillae and dentate margins, a combination of features only seen in section Acanthophyllum.
Conclusions
The present study reveals that seed characters such as shape and color as well as testa features show significant variability, and provide useful information for taxonomy of Acanthophyllum at sectional and specific ranks. Seed characters in the examined species of Acanthophyllum, support the monophyly of sections Macrostegia, Oligosperma (excluding A. caespitosum), Pleiosperma (excluding A. sordidum) and the core group of section Acanthophyllum. The exclusion of A. sordidum from section Pleiosperma and A. caespitosum from section Oligosperma, as proposed by Pirani et al. (2014), is confirmed here, suggesting that examining seed features in additional taxa would be useful in resolving infrageneric relationships. While the placement of A. caespitosum and A. sordidum in Fig. 3 may suggest that these taxa represent additional sections of Acanthophyllum, this action should be delayed until a more comprehensive study is undertaken.
Acknowledgements
We are grateful to H. Moazzeni (Ferdowsi University of Mashhad) for his assistance in field collections and valuable suggestions on the manuscript. We also wish to thank Directors and Curators of the herbaria FUMH, TARI, TMRC and TUH for permission to obtain seeds used in this study. AP appreciates financial supports provided by Ferdowsi University of Mashhad.
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