Systematics of the Mecardonia acuminata (tribe Gratioleae, Plantaginaceae) complex of southeastern USA
TABLE OF CONTENTS Page ACKNOWLEDGEMENTS iv LIST OF TABLES ix LIST OF FIGURES xi ABSTRACT OF DISSERTATION xiii LIST OF APPENDICES 140 CHAPTER I. Seed Morphology in Tribe Gratioleae 1 (Plantaginaceae) and its Taxonomic Significance Abstract 2 Introduction 4 Materials and Methods 10 Results 12 Reticulate Seed Patterns 13 Striate Seed Patterns 15 Discussion 17 Literature Cited 23 Tables 27 Figures 32
Page CHAPTER II. Genetic Variation on Inter-Simple Sequence Repeats (ISSR) Markers in the Mecardonia acuminata Complex in Southeastern USA 39 Abstract 40 Introduction 41 Materials and Methods 43 Sampling Strategy 44 ISSR Amplification 44 Data Analysis – Genetic Diversity 44 Data Analysis – Genetic relationships 45 Results 47 Genetic Variation 47 Genetic Relationships 48 Discussion 50 Genetic Diversity 50 Genetic Relationships 53 Systematic and biogeographic implications 53 Literature Cited 56 Tables 62 Figures 76
Page CHAPTER III Morphological Variation in the Mecardonia acuminata (Plantaginaceae) Complex in Southeastern USA 79 Abstract 80 Introduction 82 Materials and Methods 85 Results 89 Morphological variation within M. acuminata 89 Character correlations within M. acuminata 90 Character correlation to latitude, longitude and biogeographic region 90 Character correlation to subspecies delimitation 92 Discussion 94 Taxonomic Implications 98 Literature Cited 100 Tables 104 Figures 111 Appendices 121
LIST OF TABLES
CHAPTER I. Seed Morphology in Tribe Gratioleae (Plantaginaceae) and its Taxonomic Significance 1. Seed surface characteristics for species of tribe Gratioleae. 27 CHAPTER II. Genetic Variation based on Inter-Simple Sequence Repeats (ISSR) Analysis in the Mecardonia acuminata Complex in Southeastern USA 1. Locations of 23 populations in southeastern USA 62 2. Attributes of ISSR primers 64 3. Genetic variability at ISSR loci 65 4. Genetic differentiation, gene flow and gene variation 67 5. Measures of genetic identity coefficient 72 CHAPTER III Morphological Variation in the Mecardonia acuminata (Plantaginaceae) Complex in Southeastern USA 1. Variations among quantitative characters 104 2. Variations among qualitative characters 107
3. Correlation among morphological characters 109 4. Results of multivariate analyses 110
LIST OF FIGURES Page CHAPTER I. Seed Morphology in Tribe Gratioleae (Plantaginaceae) and its Taxonomic Significance Seed types of Mecardonia, Bacopa, and Benjaminia. 32 1. Seed types of Gratiola, Sophronanthe, Amphianthus and Scoparia 34 2. Seed types of Stemodia 36 CHAPTER II Genetic Variation based on Inter-Simple Sequence Repeats (ISSR) in the Mecardonia acuminata Complex in Southeastern USA 1. Map of eastern USA showing population locations 76 2. Neighbor-joining dendrogram 77 CHAPTER III Morphological Variation in the Mecardonia acuminata (Plantaginaceae) Complex in Southeastern USA 1. Map of distributional range of M. acuminata 111 2. Box plots illustrating variations in morphological characters 113
Page 3. Linear Regression Analysis 114 4. Canonical Discriminant Function Analysis Compared to latitude and longitude 116 5. Canonical Discriminant Function Analysis Compared to biogeographic regions 117 6. Canonical Discriminant Function Analysis Compared to subspecies 118 7. Distribution of subspecies peninsularis and and microphylla 119
DISSERTATION ABSTRACT Seed surface morphology is known to be of taxonomic significance in some plant taxa, and has been used as diagnostic features of some families, genera and species. The tribe Gratioleae (Plantaginaceae) consists of 16 genera with worldwide distribution. Similar seed surface morphologies have been observed in some members of the tribe. This study employed scanning electron microscopy (SEM) to examine detailed seed surface scultpturings of 37 species belonging to eight genera of the tribe. Sixteen seed types were identified and unique to most genera. The overall diversity of seed surface morphology observed in the tribe Gratioleae suggests extensive but taxonomically significant seed morphological variations in the tribe. Three reticulate seed types were identified for the genus Mecardonia that has three species endemic to the USA. Mecardonia acuminata, a widespread species in southeastern USA consists of at least three subspecies (acuminata, peninsularis and microphylla). Inter-simple sequence repeat markers (ISSR) were employed to elucidate the genetic variation of 23 populations in the species complex. Morphological examinations of the individuals sampled across the entire range of the species were also performed to evaluate subspecies diagnostic features and to assess the actual distributional range of each of the subspecies. Analysis of ISSR markers confirmed a widespread distribution of subspecies acuminata and identified populations with high genetic diversities occurring mainly in the southern ranges of the species. The ISSR analysis also revealed some populations of subspecies microphylla that were originally considered to be populations of subspecies acuminate. Morphological analyses also revealed possible broad historical range
distributions of subspecies peninsularis and microphylla that occurred throughout most of the range of subspecies acuminata. Clinal variations were also observed in some characters particularly leaf length which was found to increase from south to north across the distributional range. Regional biogeographic analysis of the morphological data revealed separation of individuals endemic to southern Florida.
SEED MORPHOLOGY IN TRIBE GRATIOLIEAE (PLANTAGINACEAE) AND ITS TAXONOMIC SIGNIFICANCE
Seed morphology provides important taxonomic characters that can be used to infer relationships among plant genera and species. The tribe Gratioleae is made up of terrestrial to aquatic herbs with non-alveolated (non-honeycomb) endosperm. The tribe consists of 16 genera and about 182 species distributed worldwide. Scanning electron microscopy (SEM) was employed to evaluate variation in testa surface patterns of 37 species representing eight genera in the tribe Gratioleae. Fiftteen seed types were identified based on testa surface sculpturing. Seed surfaces were mostly reticulate with radial walls of high or low relief and varied tangential wall patterns. The combination of radial wall thickenings and tangential wall patterns suggests varying seed types that are unique to most genera. Similar seed types were observed among some species of the genus Gratiola and its segregate monotypic genus Sophronanthe, and the genus Bacopa and its segregate monotypic genus Benjaminia. These observations suggest that the segregation of Sophronanthe from Gratiola and Benjaminia from Bacopa are ambiguous. The seed types observed for the genus Mecardonia also a segregate of Bacopa were significantly different from those of Bacopa. The distinct differences in seed morphology observed for these two genera support the segregation of Mecardonia from Bacopa. Seed surfaces of the genus Stemodia were either striate or reticulate. Four seed types were identified for the genus Stemodia alone, suggesting seed morphological variations in the genus. This observation indicates that the taxonomic placement of some species of the genus is ambiguous. The overall diversity of seed surface morphology observed in the tribe Gratioleae suggests extensive but taxonomically significant seed morphological
variations in the tribe. The current study also demonstrates the taxonomic significance of intricate seed surface sculpturing and the importance of employing SEM in plant systematics.
INTRODUCTION Seed surface morphology provides many useful taxonomic characters that can be used to distinguish genera and some species in the order Lamiales (Ichaso, 1978; Thieret 1954; Elisens, 1985). Evidence of the taxonomic significance of seed surface ornamentations has been documented in various plant families including the Scrophulariaceae (Thieret, 1954; Ichaso, 1978), Hydrangeaceae (Hufford, 1995) and Annonaceae (Svoma, 1998). Seed morphology can be used to infer relationships within families (Hufford, 1995; Svoma, 1998), within tribes (Elisens and Tomb, 1983, 1985; Takahashi, 1993), and within genera (Mathews and Levin, 1986; Ness 1989). For example, investigations of seed morphology have been used to support relationships within the tribes Antirrhineae of Plantaginaceae (Elisens and Tomb, 1983), and Pyroloideae of Ericaceae (Takahashi, 1993); and within the genera Cordylanthus and Orthocarpus of Orobanchaceae (Chuang and Heckard, 1972, 1983); Paulownia of Paulowniaceae (Vujicic, 1993), and Nama of Hydrophyllaceae (Bacon and Bragg,1986). Despite the reliability of seed morphology in taxonomy, it has been less commonly used than vegetative and floral characters, or nuclear and chloroplast markers (Barthlott, 1981, Albach & al., 2005). Several seed morphological variations of taxonomic significance have been identified among some members of the order Lamiales particularly in the families Plantaginaceae and Orobanchaceae (Ichaso, 1978; Thieret, 1954, 1967). Light microscopic seed examinations of four genera of tribe Antirrhineae (Plantaginaceae) endemic to Brazil by Ichaso (1978) revealed five seed types unique to those genera. Subsequent Scanning Electron Microscopy (SEM) seed examinations on representative species of all sections of that tribe by Elisens and Tomb (1983)
revealed a total of seven morphological categories based on seed shape and surface ornamentation. Examinations of seed coat ornamentations in the family Orobanchaceae revealed three seed coat types in the genus Orthocarpus as well as many species-level differences (Chuang and Heckard, 1972, 1983). Seed surface investigations of four species of the genus Agalinis were found to be taxonomically significant for species identification (Canne, 1980). Similarly, seed surface ornamentations of the genus Aureolaria (Orobanchaceae) and genus Angelonia (Plantaginaceae) were also found to be taxonomically significant for species identification (Canne, 1980; Moro & al., 2001). Previous light microscopic seed investigations of members of the tribe Gratioleae (Scrophulariaceae s.l) endemic to Central America (Thieret, 1954) revealed four main seed types including the reticulate seed type in the genera Bacopa and Lindernia, and longitudinally furrowed seed type in the genus Stemodia. Subsequent light microscopic investigations of 99 species of the family Scrophulariaceae (s.l.) that included tribe Gratioleae revealed 17 seed types in six tribes (Ichaso, 1978). In that study, six seed types were identified for tribe Gratioleae and at least 3 seed types for the genus Stemodia alone. Reticulate seed types were predominantly observed for some members of tribes Digitalideae and Buchnereae and most members of tribe Gratioleae. Thieret (1967) also observed similarities between seed characters of the genera Bacopa and Scoparia in tribe Gratioleae and Albach & al., (2005) suggested that seed characters may be useful for characterizing tribe Gratioleae.
Despite taxonomic evidence of the importance of seed surface morphology in some genera of the family Scrophulariaceae (s.l), very few detailed SEM studies have been conducted on the identification of seed types at the tribal or subtribal levels. The tribe Gratioleae as presented by Bentham and Hooker (1876) comprises 37 genera distributed worldwide particularly in North and South America, Australia and Africa. The tribe was characterized by an evoluted corolla tube, four or two stamens with two distinct anther locules, and capsules with two or four valves. One of the most important characters that distinguish the tribe is the seed morphology, which includes smooth or furrowed endosperm and longitudinal ridges with hook-like wall thickenings (reticulate) of the testa cells (Rahmanzadeh & al., 2005). Wettstein (1895) also recognized 37 genera in the tribe but excluded eight genera formerly classified by Bentham and Hooker (1987) namely, Herpestis, Microcarpaea, Mimulus, Limnophilia, Beyrichia, Bonnaya, Vandelia and Sibthorpia. Wettstein’s classification of tribe Gratioleae, however included Achetaria, Ambulia, Brythophyton, Dizygostemon, Geochorda, Ildefonsia, Lindernia, Mimetanthe, Otacanthus and Bacopa and was revised to include Herpestis (Wettstein, 1891; Albach & al., 2005). Recent molecular phylogenetic studies using two or three plastid genes by Olmstead and Reeves, (1995) and Olmstead & al., (2001) recognized 25 genera of Wettstein’s Gratioleae as part of the “Scroph II” clade, thereby excluding 12 of his genera. The tribe Gratioleae of the “New” Plantaginaceae (APG 1998; 2003; Olmstead, R. G. 2001; Oxelman & al., 2005), as proposed by Albach & al (2005), corroborated with the “Scroph II” of Olmstead and Reeves (1995).
Fischer (2004) in his recent treatment of the family Scrophulariaceae (s.l.), classified the family into 3 subfamilies; Antirrhinoideae, Gratioloideae, and Digitalidoideae. Subfamily Gratioloideae was further divided into 5 main tribes; Gratioleae, Angeloniaeae, Stemodieae, Limoselleae and Lindernieae (Fischer 2004). According to Fischer’s classification, the tribe Gratioleae is made up of terrestrial to aquatic herbs with non-alveolated (non-honeycomb) endosperm. The tribe consists of 16 genera and about 182 species that can be grouped into three subtribes; Caprarinae, Dopatrinae and Gratiolinae. Subtribe Gratiolinae is characterized by herbs that are mostly aquatic, with opposite leaves, racemose or frondose inflorescence with subrotate to 2-lipped corolla and two to four stamens (Fischer, 2004). The fruit is a capsule and seeds of most members have been reported to be mainly reticulate or striate (Fischer, 2004; Ichaso, 1978; Thieret, 1954; 1967). Subtribe Gratiolinae consists of ten genera and about 121 species with temperate and tropical America or Pantropical distribution (Fischer, 2004; Pennell, 1935, 1946; Thieret, 1954). The ten genera belonging to this subtribe are Bacopa and Mecardonia (formerly Herpestis), Amphianthus, Gratiola, Sophronanthe, Benjaminia, Scoparia, Boelkea, Maeviella and Braunblequetia (Fischer, 2004). In a recent molecular phylogenetic analysis of the enlarged family Plantaginaceae using nuclear and plastid DNA regions (Albach & al., 2005), the genera Bacopa, Mecardonia, Scorparia, Gratiola, Otacanthus and Stemodia formed a well supported clade (Bootstrap = 90%). The first four genera of this lineage are also members of subtribe Gratiolineae, therefore partly supporting Fischer’s classification of the subtribe. Similarly, a phylogenetic study of members of the order Lamiales using four plastid DNA sequences found a well supported clade
(bootstrap = 98%) for the genera Gratiola, Scoparia, Mecardonia and Stemodia (Oxelman & al., 2005). A previous molecular phylogenetic study of the family Scrophulariaceae (s.l) found a well supported clade (Bootstrap = 100%) comprising Bacopa, Gratiola and Amphianthus (Olmstead et al., 2001). Scanning electron microscopy investigations of seed coat ornamentations provide insights into intricate microsculpturing patterns on the radial and tangential walls of the seed coat cells Canne, 1979, 1980; Chang and Heckard, 1972, 1983; Vujicic, 1993; Juan & al., 1994; Moro et al., 2001). Although previous seed surface studies of some members of Plantaginaceae by Thieret (1954; 1967) and Ichaso (1978) have suggested that reticulate seed type is common in tribe Gratioleae, particularly in subtribe Gratiolinae, and it has been observed that there is some similarity between seed characters of Bacopa and Scoparia (Thieret, 1967), no detailed SEM observations of seed surface ornamentations have been conducted at the tribal or subtribal level. The present paper presents SEM investigations of seed morphology in the subtribe Gratiolinae (Fischer, 2004). The investigations include species of eight of the ten genera classified under the subtribe as well as some species of the genus Stemodia (Table 1). The genus Stemodia was included in the current study since previous morphological studies (Bentham and Hooker, 1876; Wettstein, 1895; Small, 1913; Bigazzi, 1993) and recent molecular phylogenetic studies of the enlarged family Plantaginaceae (Olmstead & al., 2001; Albach & al., 2005; Oxelman & al., 2005) have found the genus to be closely related to some members of subtribe Gratiolinae, and the group was referred to as the ‘core” Gratioleae in Albach et al., 2005). In Fischer’s treatment of the Scrophulariaceae, the genus Stemodia is
classified under tribe Stemodieae (Fischer, 2004) The relevance of this investigation is therefore, to identify similarities or differences in seed surface ornamentations among genera of the subtribe Gratioliinae and the genus Stemodia, and to identify new and additional morphological characters that can be used in delimiting the group. Examinations of microsculpturing of the seed coats of species of the genera will serve as additional taxonomic evidence for the tribe Gratioleae as a whole.
MATERIALS AND METHODS Mature whole seeds were removed from herbarium specimens obtained from the Missouri Botanical Garden (MO), the Botanical Research Institute of Texas (BRIT), Vanderbilt University (VDB), New York Botanical Garden (NY), and the University of Oklahoma (OKL). Samples were obtained from 41 species representing eight genera considered closely related in taxonomic treatments of subtribe Gratiolinae in tribe Gratioleae (Fischer 2004) or indicated as members of a monophyletic clade in molecular phylogenetic analyses (Albach & al., 2005; Rahmanzadeh & al., 2005; Estes and Small in press, unpublished data) (Table 1). Eighteen species were sampled for Bacopa, nine species for the genus Mecardonia, six species for Stemodia, three species for Gratiola, two species for Scoparia and one species each for the monotypic genera Sophronanthe, Benjaminia and Amphianthus. Samples of species of Bacopa were obtained for five of six sections (Pennell, 1946): Bacopa Wettstein, Bramia (Lamarck) Wettstein, Chaetodiscus (Bentham) Wettstein, Herpestis (C. F. Gaertner), and Mella (Vandelli) Wettstein. Samples of section Silvinula (Pennell) were not included in this study. Seeds from multiple specimens were observed initially under the compound microscope and representative seeds were selected from one herbarium sheet to represent each species. One to four seeds per species were mounted on double-sided carbon tape affixed to aluminum SEM stubs. Specimens were sputter-coated with approximately 200 Å of gold/palladium. Seeds were examined on a JEOL-880 SEM operating at 15 kV and images digitally captured using IXRF/EDS system. Images
were prepared, and plates were assembled using Adobe Photoshop version 7.0. A list of specimen collections examined is provided in the Appendix 1-1.
RESULTS Among species examined, seeds are numerous per capsule and range in size from 0.4 mm (Mecardonia flagellaris) to 1.1 mm (Amphianthus pusillus). Seed shapes are ellipsoidal, cylindriodal or ovoid, and vary depending on seed packaging in the capsule. A cross section of Mecardonia acuminata seeds shows that the seed coat consists of two layers of cells. The inner layer is made up of small and rectangular cells whereas the outer layer comprises of large cells. The radial cell walls of the outermost layer the epidermis, are thickened and project into ridges to form a reticulate pattern (not shown). Fifteen seed patterns were identified (Table 1). Thirteen reticulate and two striate seed patterns were described based on general seed surface pattern, variation in the ornamentation of the tangential walls, and the relative height and ornamentation of the radial walls. Reticulate seeds had a surface pattern characterized by a reticulum outlined by elongated radial walls against a tangential surface without grooves or lines. In contrast, striate seeds had several longitudinal grooves present on the seed surface. Tangential wall ornamentation varied in both reticulate and striate seeds, with the structural range characterized as smooth, alveolate, corrugate, nodulate, papillate, rugulate, or verrucate. Radial walls were assessed subjectively with either ‘high’ or ‘low’ relative height, whereas radial wall ornamentation patterns were described as smooth, mammilate, or nodulate. Species of Mecardonia exhibited three reticulate seed patterns (1, 2, 3) that differed in the ornamentation pattern of the radial walls. Patterns 1 and 3 were unique to Mecardonia, although pattern 2 was found also in Amphianthus pusillus. Species
of Bacopa were characterized by five reticulate patterns (4, 5, 6, 7, 8), which differed primarily in ornamentation of the tangential walls that were either alveolate, papillate, reticulate, or verrucate. Seeds of Bacopa generally had low radial walls, except for B. crenata. Reticulate pattern 5 was the most common pattern in Bacopa (eight species) and also was observed in seeds of Benjaminia reflexa (= Bacopa reflexa (Benth.) Loefgr. & Edwall). Reticulate patterns 9 and 10 were confined to Gratiola and differed in height of the radial walls and tangential wall ornamentation. Reticulate 10 seeds were present also in the segregate Sophronanthe hispida (= Gratiola hispida Pollard). Whereas both species examined in the genus Scoparia had seeds characterized as Reticulate 11, four unique seed patterns were observed in Stemodia. Two reticulate patterns (12, 13) and two striate patterns (1 2) were observed among seeds of six species of Stemodia. Striate seeds were observed only in the genus Stemodia. The 15 primary seed patterns are described below. Reticulate Seed Patterns. Reticulate seeds characterize the Gratioleae as usually defined (Thieret 1954, Dathan 1995, Fischer 2004, Olmstead et al. 2005, Rahmanzadeh et al. 2005), although Stemodia with both reticulate and striate seeds has been placed in the Stemoideae by Fischer (2004) and Rahmanzadeh et al. (2005). Reticulate pattern 1 (Figs. 1A, 1B) was characterized by smooth tangential walls and high radial walls with a mammilate ornamentation. Reticulate I seeds were observed only in the genus Mecardonia in M. dianthera, M. procumbens, M. vandelloides and M. veronicaefolia. Reticulate Pattern 2 (Figs. 2G, 2H) was characterized by smooth tangential and radial wall ornamentation and high radial walls. Reticulate II seeds were
confined to Amphianthus and three species of Mecardonia: M. acuminata, M. montevidensis, and M. tenella. Reticulate Pattern 3 (Figs. 1C, 1D) had seeds with smooth tangential walls and high radial walls with nodulate ornamentation. Reticulate III seeds only were observed for M. flagellaris. Reticulate Pattern 4 (Figs. 1E, 1F) was characterized by alveolate tangential walls with smooth radial walls of comparatively low relief. This seed pattern was observed only in B. axillaris in Section Mella and B. egensis, B. rotundifolia, B. salzamanii and B. stragula in Section Herpestis. Reticulate Pattern 5 (Figs. 1G, 1H, 1K, 1L) seeds had papillate tangential walls and smooth radial walls with low relief. It was the most common seed type in Bacopa and characterized the seeds of eight species in two sections: B. aquatica, B. bacopoides, B. decumbens, B. floribunda, B. gratiloides, B. lacertosa, B. laxiflora (all of Section Mella), and B. monnieri (Section Bramia). Reticulate V seeds also occurred in Benjaminia reflexa. Reticulate Pattern 6 (not shown) seeds were observed only in Bacopa crenata of Section Mella. The seed coat had papillate tangential walls and smooth radial walls of high relief. Reticulate Pattern 7 (not shown) seeds also were observed only in Bacopa in three species of Section Mella (B. auriculata, B. bracteolata, B. sessiflora) and B. caroliniana of Section Chaetodiscus. Reticulate VII seeds had verrucate tangential walls with smooth radial walls of low relief.
Reticulate Pattern 8 (Figs. 1I, 1J) was characterized by reticulate tangential walls and low radial walls with smooth ornamentation. Reticulate VIII seeds were observed only in B. egensis of Section Herpestis. Reticulate Pattern 9 (Figs. 2C, 2D) had seeds with verrucate tangential walls and smooth radial walls of high relief. This seed pattern was observed only in Gratiola aurea and G. neglecta. Reticulate Pattern 10 (Figs. 2A, 2B, 2E, 2F) was characterized by corrugate tangential walls and low radial walls with a smooth ornamentation pattern. Reticulate X seeds were observed in Gratiola pilosa and in the segregate genus Sophronanthe (S. hisida). Reticulate Pattern 11 (Figs. 2I, 2J, 2K, 2L) seeds had rugulate tangential walls with smooth radial walls of low relief. Seeds with this pattern were observed for the two species examined for Scoparia, S. dulcis and S. montevidensis. Reticulate Pattern 12 (Figs. 3E, 3F) had seeds characterized by verrucate tangential walls and low radial walls with a nodulate ornamentation pattern. Reticulate XII seeds were observed in Stemodia durantifolia and S. lanceolata. Reticulate Pattern 13 (Figs. 3G, 3H) seeds were similar to Reticulate XII seeds with verrucate tangential walls and nodulate radial walls, but differed from Reticulate XII by high radial walls. Seeds with this pattern only were observed in Stemodia schottii and S. stricta. Striate Seed Patterns. Striate seeds were observed only in two species of the genus Stemodia. Two patterns were observed and differed in tangential wall ornamentation and radial wall height and ornamentation pattern.
Striate Pattern 1 (Figs. 3C, 3D) seeds combined longitudinal grooves and reticulate patterns on the seed surface. The tangential walls were verrucate and the radial walls had a low relief with a smooth ornamentation pattern. Striate I seeds were observed only in Stemodia suffructicosa. Striate Pattern 2 (Figs. 3A, 3B) seeds were characterized by longitudinal grooves and a surface sculpturing pattern described as nodulate. Reticulations are not apparent or similar in appearance to those described for the reticulate seed patterns. Striate II seeds were observed only in Stemodia verticillata.
DISCUSSION The identification of 12 reticulate seed types for the nine genera investigated demonstrates extensive variation in reticulate seeds. Reticulate testas observed for all species of subtribe Gratiolinae also support the monophyly of the group (Thieret, 1954; Fischer, 2004; Albach & al., 2005). Most of the large genera had two or more seed types that were unique to the genus. These unique reticulate seed types provide further taxonomic evidence in support of the monophyly of each of the genera, Scoparia Bacopa and Mecardonia (Rossow, 1987; Pennell, 1946). One seed type (XI), was identified for the two species of Scoparia investigated. Although this genus had limited sampling, the identification of seed type XI, can be considered a representation of the seed type for the genus. Seven seed types were identified for the genus Bacopa which has about 60 species distributed worldwide. The seed types of Bacopa were all unique to the genus, but not unique to any particular section thereby indicating that, either seed characters are not useful in section characterization or the taxonomic circumscription of the sections may be ambiguous (Pennell, 1946). None of the Bacopa seed types was identified in the genus Mecardonia which is a segregate of Bacopa (Pennell, 1946). Despite the variation of seed types in this genus, four of the seed types were not identified in any other genus investigated. Seed type IV was however observed in the monotypic genus Benjaminia (Fig. 1K, L), a segregate of Bacopa (Bentham, 1873), section Chaetodiscus (Pennell, 1946). Seed type V (papillate tangential walls and smooth radial walls with low relief) was identified in eight of the eighteen species of Bacopa examined (Table 1), and was the most common seed type observed for the genus. Benjaminia reflexa has long been
considered congeneric with Bacopa but differs in its dissected leaves and slightly connate subequal clayx lobes (D’Arcy, 1979). The shared seed type V observed between Benjaminia and some members of Bacopa provides additional evidence in support of previous classification of this genus as a species of Bacopa (Bentham, 1873; Pennell, 1946). The three seed types I, II and III observed for the genus Mecardonia, also a segregate of Bacopa (Pennell, 1923), were not observed in any species of Bacopa s.s.. The tangential walls of all these seed types are smooth, whereas those of Bacopa are sculptured (Table 1; Fig. 1E – J). These unique seed types of Mecardonia support the segregation of the genus from Bacopa (Pennell, 1946; Rossow, 1987). Seed type II of Mecardonia was also observed in the genus Amphianthus. However, the seeds of Amphianthus are much bigger (1.1 mm long) - about double the size of Mecardonia seeds (0.4 mm – 0.6 mm). The shared seed type between Amphianthus and some members of Mecardonia suggests that these two genera may be closely related than originally thought. Seed examination of the genus Gratiola, revealed 2 main seed types, IX (G. aurea and G. neglecta) and X (G. pilosa). Seed type X was similar to that observed for the genus Sophronanthe except that, the corrugate tangential wall patterns on S. hispida were not as closely spaced as observed in G. pilosa. The taxonomic placement of G. pilosa, was once in the genus Sophronanthe and then Tragiola (Small, 1933). The presence of similar seed types observed for the two taxa suggests close relatedness between these two taxa. Phylogenetic analysis of members of the genus Gratiola using ndhF gene sequences revealed that G. pilosa is sister to S. hispida (Estes and Small, unpublished data). Striate seed types (Thieret, 1979) were
not identified for any of the genera examined under subtribe Gratiolinae except for the genus Stemodia (tribe Gratioleae/Stemodieae). The identification of both reticulate and striate seed types for the genus Stemodia demonstrates variations in the seed morphology for that genus and corroborates with previous seed investigations of the genus (Thieret, 1954, Ichaso, 1978, Fischer, 2004). The taxonomic placement of the genus in the tribe Gratioleae remains ambiguous (Fischer, 2004, Albach & al., 2005). Whereas most molecular and morphological studies have included Stemodia under the tribe Gratioleae (Albach & al., 2005; Oxelman & al., 2005; Thieret, 1978), some morphological studies have included the genus in the tribe Stemodieae (Fischer, 2004). Both Gratioleae and Stemodieae are classified under the subfamily Gratioloideae (Fischer, 2004; Rahmanzadeh & al., 2005). Previous Stemodia seed investigations by Thieret (1978) revealed three seed types; reticulate, striate and granulate. In the current study, two types of reticulate seeds (XII and XIII) and two types of striate seeds (Striate I and II) were identified (Fig. 3).The presence of five seed types in six of the Stemodia species investigated suggests variation in seed morphology and concurs with its uncertain taxonomic placement in the family. These seed observations raise questions on the infrageneric relationships, and suggest that the genus may be polyphyletic. In an attempt to determine the phylogenetic relationships among members of the newly segregated Plantaginaceae, Albach & al., (2005) employed nuclear and plastid markers to establish well supported clades within the family. One of these clades was the Gratioleae clade of which Gratiola, Bacopa, Mecardonia, Scoparia, Stemodia and Otacanthus were referred to as the ‘core’ Gratioleae. Previous phylogenetic studies of