Rolf Y. Berg 2 - American Journal of Botany

American Journal of Botany 96(3): 565–579. 2009. 

Considerable disagreement has existed with regard to generic 

limits and generic relationships within the former Hydrophyllaceae, 

now part of the Boraginaceae (APG II, 2003). Embryological 

information in its broadest sense is complementary to 

molecular phylogenetics and of particular value in delimiting 

genera because, as a rule, the embryological characters of the 

species within a genus are constant ( “ Cave ’ s law ” ; Cave, 1953, 

p. 140). Also, within the tribe Hydrophylleae, seed characters 

are extraordinarily important taxonomically, but the lack of ontogenetic 

studies has resulted in confusing terminology and 

much misunderstanding. This study aims at verifying the taxonomic 

treatment of Nemophila by means of embryological and 

seed ontogenetical data. In addition, from a theoretical point of 

view, Nemophila is a particularly good test example of the validity 

of Cave ’ s law: Two species have been excluded. The remaining 

11 species fall into two morphologically different but 

taxonomically unrecognized groups. The size of the genus, 

11 – 13 species, seems appropriate. Materials are readily obtained: 

All species are annuals and easily grown from seed, and 

all species, except two, grow wild in California and Oregon. 

1 Received for publication 25 June 2008; revision accepted 24 October 2008. 

This study initially was supported by the United States International 

Cooperation Administration through the E.P.A.-151 Project. Later, support 

was received from the Norwegian Research Council for Science and the 

Humanities. The author is grateful to the Department of Botany, University 

of California, at Davis and at Berkeley, for laboratory accommodation 

(Davis 1954, 1992 – 1993; Berkeley, 1955, 1980); to the late M. S. Cave, L. 

Constance, K. Esau, and E. M. Gifford for sharing their time and wide 

knowledge; to T. Berg, for assisting in the fi eld, for doing the microtechnical 

work, and for inking all drawings; to T. I. Chuang for providing the SEM 

photographs, to E. Timdal for IT help and producing Figs. 52 – 64 from 

microphotographs, to E. G. Cutter for reading the manuscript and offering 

important and helpful comments, and to the associate editor, two anonymous 

reviewers, and B. E. Hazen for suggestions that improved the manuscript. 

2 E-mail: r.y.berg@nmh.uio.no 

doi:10.3732/ajb.0800208 

E MBRYO SAC, ENDOSPERM, AND SEED OF N EMOPHILA 

(BORAGINACEAE) RELATIVE TO TAXONOMY, WITH A REMARK ON 

EMBRYOGENY IN P HOLISTOMA 1 

Rolf Y. Berg 2 

Department of Botany, Natural History Museum, University of Oslo, P.O. Box 1172 Blindern, 0318 Oslo, Norway 

Studies on embryology and seed morphology are complementary to molecular phylogenetics and of special value at the genus 

level. This paper discusses the delimitation and evolutionary relationships of genera within the tribe Hydrophylleae of the Boraginaceae. 

The seven Nemophila species characterized by a conspicuous seed appendage are similar in embryology and seed structure. 

The ovule is tenuinucellate and unitegmic with a meristematic tapetum. The embryo sac penetrating the nucellar apex is of 

the Polygonum type, has short-lived antipodal cells, and an embryo sac haustorium. The endosperm is cellular, producing two 

terminal endosperm haustoria, of which the chalazal has a lateral branch. Embryogeny is of the Chenopodiad type (as in Pholistoma 

). The seed coat is formed from the small-celled inner epidermis of the integument. The large-celled outer epidermis of the 

integument disintegrates into scattered cells. Seed pits evolve from irregularly placed inner epidermal cells of the integument. The 

chalazal part of the ovule produces a cucullus, that functions as an ant-attracting elaiosome. Those species of Nemophila with a 

conspicuous cucullus form a natural genus. Nemophila is most closely related to Pholistoma . The integumentary seed pits of Nemophila 

might have evolved from ovular seed pits similar to those in Pholistoma. 

Key words: Boraginaceae; Hydrophyllaceae; myrmecochory; Nemophila; Pholistoma; plant embryology; seed development; 

taxonomy. 

565 

However, from a practical point of view, Nemophila proved far 

from ideal as a test case of Cave ’ s law, as described later in the 

methods. The practical diffi culties caused this investigation to 

be abandoned several times over the years. 

The fi rst purpose of this study, then, is to increase our knowledge 

of Nemophila . The genus is poorly studied with regard to 

ovule, embryo sac, embryo, and endosperm. Nemophila menziesii 

Hook. & Arn. is the only reasonably well-known species 

( Hofmeister, 1858 ; Svensson, 1925 ; Cr é t é , 1947 ). A few scattered 

data are available also for N. phacelioides Nutt. ex Barton 

(J ö nsson, 1881) and N. maculata Lindley ( Svensson, 1925 ). 

Early stages of endosperm development are known from fi ve 

species ( DiFulvio, 1987 ). 

The second purpose is to study its seed. Constance (1941) , in 

his taxonomic delimitation of Nemophila , placed special emphasis 

upon the following seed characteristics: (1) Seed appendage 

or “ cucullus. ” Ecucullate species were removed to 

other genera, and within Nemophila , species were keyed into 

those having a “ conspicuous cucullus ” and those having a “ reduced 

cucullus. ” (2) Seed pits. Only species with pitted seeds 

were included in Nemophila . Within the genus, species were 

keyed into those having uniform pits arranged in regular rows 

and those having unequal pits irregularly arranged. (3) Seed 

coat pattern. A fi nely reticulate seed coat is characteristic of 

Nemophila . All related genera have seeds with coarsely reticulate 

or alveolate coats. 

Later, Chuang and Constance (1992) treated seed characters 

in Nemophila and related genera in more detail, trying to explain 

how these distinctive seed characters do arise. Here, I 

verify, illustrate, and supplement earlier data on the ontogeny, 

anatomy, and morphology of these seed characteristics. I also 

offer some thoughts on their function and evolution. 

Brand (1913) grouped the genera of the Hydrophyllaceae 

into three tribes: Hydrophylleae, Phacelieae, and Hydroleae. 

Constance (1939a) recognized fi ve genera within tribe Hydrophylleae: 

Hydrophyllum , Pholistoma , Ellisia , Nemophila , and 

Eucrypta. Pholistoma was constructed as a combination of

566 American Journal of Botany [Vol. 96 

three species removed from Ellisia and Nemophila ( Constance, 

1939b ). Berg (1985) presented embryological evidence in support 

of this new generic unit. Di Fulvio De Basso (1990) argued 

for the removal of Eucrypta from Hydrophylleae to the Phacelieae 

on the basis of endosperm characteristics, a view that was 

later supported by Chuang and Constance (1992) . Molecular 

data provided by Ferguson (1999, p. 263) do support a Hydrophylleae 

of fi ve genera, including Eucrypta ( chrysanthemifolia 

), as originally outlined by Constance (1939a) . Ferguson ’ s 

results for E. micrantha , however, are ambiguous. A third purpose 

of this study is to evaluate the new information presented 

on embryology and seed with regard to possible taxonomic and 

evolutionary implications. 

As mentioned, Constance (1941) keyed his Nemophila species 

into those having a reduced and those having a conspicuous 

cucullus. Embryological and seed anatomical characters 

strengthen the difference between those two groups of species. 

For technical reasons, the embryology and seed anatomy of the 

“ reduced cucullus species ” will become treated in a separate 

paper, now in preparation. The fi nal purpose of this study, to 

present a test of Cave ’ s law, mostly will be postponed until that 

paper. Here, all of the seven “ conspicuous cucullus ” species of 

Constance (1941) are treated, i.e., N. heterophylla , N. maculata 

, N. menziesii , N. parvifl ora , N. pedunculata , N. pulchella , 

and N. spatulata. A few preliminary observations were included 

in a speech given at The Norwegian Academy of Science and 

Letters in 1982 ( Berg, 1984 ). 

MATERIALS AND METHODS 

Practically all fi xations for light microscopy were made in 

the fi eld in California, USA (Appendix 1). A few were made 

from plants cultivated, in Davis, California, or in Oslo, Norway, 

from fi eld-collected seeds. SEM micrographs were made 

from herbarium material (Appendix 1). 

Material for light microscopy was fi xed in Belling ’ s modifi 

ed Navashin fl uid (Johansen, 1940), dehydrated after two days 

to three months with tertiary butyl alcohol, embedded in paraffi 

n, sectioned on a rotary microtome at 10 – 20 µ m, and stained 

with safranin and fast green. The material had to be divided into 

very small pieces to secure proper fi xation. As in Pholistoma 

( Berg, 1985 ), longitudinal sections of ovules were exceedingly 

diffi cult to obtain because of the irregular arrangement of ovules 

within the ovary. Approximately 3100 ovaries, fruits, and seeds, 

representing nearly 20 000 ovules were sampled and embedded 

in paraffi n, and the following approximate numbers of ovules/ 

seeds were sectioned and studied: Nemophila heterophylla , 

175; N. maculata , 900; N. menziesii , 270; N. parvifl ora , 110; N. 

parvifl ora var. austinae , 25; N. pedunculata , 265; N. pulchella , 

70; N. pulchella var. fremontii , 10; and N. spatulata , 220. Embedded 

seeds and older ovaries were softened for several days 

before sectioning, in a mixture of 10 parts hydrofl uoric acid, 10 

parts glycerin, and 80 parts 95% ethanol. Light micrographs 

were taken with a Zeiss Axioplan 2 light microscope equipped 

with an AxioCam HRc camera. For SEM, the material was 

treated as described by Chuang and Constance (1992) . 

RESULTS 

Ovule — The ovule is basically similar in all Nemophila species 

here treated. It is tenuinucellate and unitegmic. Shortly af- 

ter initiation, the ovule begins to curve, and the fi rst of the 

integumentary cells appear on its convex side ( Fig. 1 ). As the 

single integument grows up about the nucellus, the ovule continues 

to curve, through pronounced growth in the chalazal region, 

becoming anatropous before fertilization ( Figs. 16, 21, 

27, 29, 52 ). A provascular strand differentiates from the placenta 

toward the chalaza ( Figs. 16, 27 ). The cells of this strand 

mostly remain parenchymatic until the ovule is fully developed 

( Fig. 29 ), but in N. parvifl ora , at least, a few sieve tubes appear. 

The vascular strand is not strengthened after fertilization, but is 

destroyed in part by the activity of the lateral endosperm haustorium 

( Fig. 34 ). 

The young nucellus consists of a unilayered epidermis over 

a core of longitudinal cell rows, about two cells in width 

( Figs. 27, 53 ). The nucellus stays small. It also is short-lived. 

Its apex is penetrated by the embryo sac approximately at the 

four-nucleate embryo sac stage ( Figs. 26, 54, 55 ). Subsequently, 

it becomes compressed and resorbed by the enlarging embryo 

sac ( Figs. 18, 28 ) and its haustorium ( Fig. 6 ). At the time of 

fertilization, part of the nucellar epidermis may still remain 

( Fig. 30 ). More often, only crushed cell remnants occur below 

the embryo sac haustorium. These might be of nucellar origin 

( Fig. 23 ) or alternatively be derived from integument/chalaza 

( Fig. 29 ). As a rule, recognizable nucellar remnants are absent 

from the ovule at the time of fertilization. 

The integument is three layers thick at the apex and four layers 

thick at the base shortly after inception ( Figs. 2, 3, 27 ). It 

grows rapidly in length and closes above the nucellus about the 

time the functional megaspore develops, sometimes a little later 

( Figs. 16, 17, 52 ). Adjacent to the nucellus, the inner integumentary 

epidermis develops into a tapetum (endothelium). The 

integumentary epidermal cells in this region elongate transversely 

with respect to the longitudinal axis of the ovule and, 

simultaneously, undergo intense anticlinal divisions ( Figs. 2 – 5, 

15 – 18, 22, 27, 28, 52 – 54 ). The result is a tissue of closely 

packed, transversely oriented, plate-shaped cells rich in cytoplasm, 

forming a cylinder about the nucellus ( Fig. 55 ). However, 

as the short-lived nucellus disappears, the tapetum cylinder 

comes to be in direct contact with the embryo sac over most of 

its inner surface ( Figs. 6, 23, 29, 56 ). The tapetum acts as an 

integumentary meristem, cutting off cells toward its periphery 

through periclinal divisions ( Figs. 18, 19, 54 ) and multiplying 

longitudinally, through anticlinal divisions. This meristematic 

activity lasts approximately from the time the functional megaspore 

develops ( Fig. 16 ) to shortly before the zygote embeds 

itself into the endosperm proper ( Figs. 9, 34, 37, 58 ). Thereafter, 

the tapetum reverts to a normal epidermis, its cells becoming 

isodiametric in shape and dividing only transversely when 

necessitated by epidermal enlargement ( Figs. 35, 38, 41, 59 ). 

The tapetum derivatives develop into integumentary parenchyma 

cells, which contribute notably to the thickness of the 

integument ( Figs. 23, 28 ). In some sections, the tapetumderived 

parenchyma cells form radial rows extending outward 

from a tapetum cell ( Fig. 29 ), thus indicating their origin. Most 

often, however, parenchyma derivatives of the tapetum and 

parenchyma derivatives of the middle layers cannot be 

separated. 

The cells of the outer integumentary epidermis are relatively 

large at the time of fertilization, more or less isodiametric, with 

a large vacuole located toward the ovule interior and with most 

of the cytoplasm and the nucleus located more or less toward 

the ovule surface ( Figs. 7, 9 ). The outer cell walls are somewhat 

thickened.

March 2009] 

Embryo sac — In all seven species, the embryo sac is of 

the Polygonum type. A single hypodermal archesporial cell 

( Figs. 1, 14, 21 ) functions directly as a megaspore mother cell 

( Figs. 2, 27 ). Meiosis ( Fig. 3 ) results in a linear tetrad of megaspores 

( Fig. 15 ). The three megaspores closest to the micropyle 

normally degenerate, the chalazal megaspore giving rise to 

the embryo sac ( Figs. 4, 16, 22, 52, 53 ). The mononucleate embryo 

sac has its nucleus centrally located, normally with one 

vacuole toward the micropyle and one toward the chalaza 

( Fig. 4 ). Occasionally, after the fi rst nuclear division in the embryo 

sac, the two sister nuclei can be observed in the middle of 

the sac ( Fig. 25 ). However, this stage seems very brief. Apparently, 

the two sister nuclei rapidly move one toward each end of 

the sac, while a vacuole develops in the sac ’ s central part 

( Figs. 17, 54 ). A nuclear division at each end produces the fournucleate 

embryo sac ( Figs. 26, 55 ) which, subsequently, through 

a last synchronous division of all its nuclei, becomes the eightnucleate 

embryo sac, with its four micropylar and four chalazal 

nuclei separated by a large, central vacuole ( Fig. 18 ). After the 

walls have been laid down in the usual manner, the mature embryo 

sac consists of three micropylar cells, forming the egg apparatus, 

three antipodal cells at the chalazal end, and two polar 

nuclei ( Fig. 28 ). The polar nuclei migrate toward each other and 

meet in the micropylar half of the sac ( Figs. 29, 56, 57 ). They 

do not fuse, but press against each other, touching not only at a 

point, but over a considerable area. When mature, each synergid 

has an apical vacuole ( Fig. 57 ), while the egg has an apical 

nucleus ( Fig. 56 ). 

Already in the bud stage, the chalazal end of the embryo sac 

develops into an embryo sac haustorium, which penetrates into 

the chalaza below the tapetum. At fi rst, the degenerating antipodal 

cells are bypassed by the embryo sac haustorium ( Fig. 6 ). 

Soon, however, the antipodal cells are resorbed by this “ aggressive 

” part of the embryo sac ( Fig. 29 ). No identifi able traces of 

antipodal cells are left at the time of fertilization ( Figs. 19, 23, 

56 ). The mature embryo sac possesses many small and large 

vacuoles, and its cytoplasm contains numerous small globules 

( Figs. 19, 29 ), presumed to be starch ( Schnarf, 1931, p. 167). 

Fertilization and endosperm — All species reproduce sexually. 

Double fertilization and triple fusion were observed in Nemophila 

pulchella ( Fig. 23 ). At the time of triple fusion, the 

polar nuclei are located near the chalazal end of the embryo sac, 

immediately above the embryo sac haustorium. The triple fusion 

is initiated as the male gamete joins the upper polar nucleus. 

Fertilization occurs rapidly, soon after pollination before 

the fl ower wilts. 

All species are similar with regard to endosperm formation: 

The endosperm is cellular. Its development is initiated immediately 

after fertilization, while the fl ower is still fresh, by division 

of the primary endosperm nucleus ( Figs. 24, 30 ). This 

nuclear division is followed by the formation of a transverse 

wall, more or less across the middle of the primary endosperm 

cell, well above what was the haustorial part of the embryo sac 

( Figs. 7, 31 ). A synchronous division of the two endosperm nuclei 

( Fig. 20 ) is again followed by transverse wall formation. 

The result is a longitudinal row of four cells ( Figs. 32, 58 ). The 

uppermost and lowermost cells of this row do not divide any 

further but differentiate into mononucleate endosperm haustoria, 

one micropylar and one chalazal, while the two middle cells 

of the row give rise to the endosperm proper. These two cells 

divide synchronously ( Fig. 33 ) by longitudinal walls. The result 

is four endosperm cells proper arranged in two two-celled tiers 

Berg — Embryology and seed of NEMOPHILA 

567 

( Fig. 36 ). The four endosperm proper cells then divide synchronously 

and longitudinally to produce eight cells in two fourcelled 

tiers ( Figs. 8, 9, 37 ). Sometimes the cells of the tier 

toward the micropyle again divide longitudinally, while some 

or all of the cells of the tier toward the chalaza divide transversely 

( Fig. 11 ). Sometimes the fi nal synchronous division is 

longitudinal in all cells, producing 16 endosperm cells, arranged 

in two eight-celled tiers ( Fig. 34 ). Less synchronous cell divisions 

then follow in more diverse directions, to produce a rapidly 

growing multicellular endosperm proper organized in 

several irregular tiers ( Figs. 35, 38 ). 

In the very beginning, the small globules, presumed to be 

starch, from the central cell of the embryo sac persist within the 

endosperm ( Figs. 24, 30, 31 ). The globules disappear as soon as 

the endosperm proper begins to form. Typically, endosperm 

proper cells are large in volume and poor in cytoplasm for as 

long as the young seed enlarges. Richest in cytoplasm during 

seed enlargement are the smaller, rapidly dividing cells along 

the periphery of the endosperm ( Figs. 41, 47 ). 

The micropylar endosperm haustorium extends around the 

zygote and grows with the seed, but does not expand markedly 

in relative size ( Figs. 34, 41, 48 ). It apparently tries to penetrate 

the tapetum just above the endosperm proper, when the endosperm 

proper consists of four to eight cells (Figs. 8, 9 ). The 

attack normally occurs at two opposite points simultaneously. 

A few tapetum cells may become destroyed ( Fig. 10 ), but the 

attack seems to be aborted and any damage to the tapetum is 

repaired. A permanent lateral branch from the micropylar 

endosperm haustorium into the surrounding integumentary 

parenchyma was never observed. However, cells of the integumentary 

parenchyma in this region become destroyed and, 

presumably resorbed, through the micropylar endosperm haustorium 

activity ( Fig. 10 ). For some time, “ scar tissue ” in the 

form of groups of old, collapsed parenchyma cells remains in 

the neighborhood of the micropylar endosperm haustorium, 

pressed together among the new parenchyma cells to the outside 

of the regenerated inner epidermis of the integument ( Figs. 

35, 41, 44 ). The micropylar endosperm haustorium remains 

mononucleate. With age, the nucleus enlarges, gradually becoming 

somewhat hypertrophied ( Figs. 34, 44 ). The micropylar 

endosperm haustorium is remarkably persistent, remaining 

a recognizable structure at least until after the globular embryo 

stage ( Figs. 39, 48, 60 ). 

The chalazal endosperm haustorium is a continuation of the 

embryo sac haustorium. After fertilization, this haustorium expands 

aggressively, often in all directions and over a large area 

( Figs. 7, 9, 30 – 35 ), increasing considerably in relative size. 

This haustorium, too, remains mononucleate for a while, but 

soon its nucleus migrates into a lateral outgrowth. Most of its 

volume is taken up by a large central vacuole. Its cytoplasm is 

concentrated about the nucleus and along the walls. A leftover, 

in the form of a small cavity, may still remain within the young 

seed at the globular embryo stage ( Fig. 39 ). The most conspicuous 

feature of the chalazal endosperm haustorium is that it produces 

a lateral outgrowth, the so-called lateral endosperm 

haustorium. 

In most species, the initiation of the lateral endosperm haustorium 

occurs when the endosperm proper is at the four- to 

eight-celled stage. At that time, the chalazal haustorium nucleus 

is located in dense cytoplasm close to the endosperm proper, on 

the side of the haustorium that faces the placenta ( Fig. 36 ). A 

bulge, formed from the chalazal haustorium adjacent to the 

nucleus, extends through the tapetum and into the surrounding


Figs. 1 – 13. Longitudinal sections of ovule, nucellus, young seed, and proembryo of Nemophila parvifl ora. 1. Very young ovule showing archesporial 

cell and initiation of integument. Bar (also for Figs. 1 – 7, 9 – 13) = 50 µ m. 2. Ovule from young bud showing prominent chalaza and small nucellus with 

megaspore mother cell surrounded by single integument. 3. As in Fig. 2, but fi rst meiotic division in megaspore mother cell. 4 . Nucellus surrounded by 

young tapetum, showing three degenerating micropylar megaspores and germinating chalazal megaspore. 5. Integument and nucellus showing remnants of 

three micropylar megaspores and two-nucleate embryo sac. 6. Ovule from bud just before opening, showing closed integument, with large-celled outer

March 2009] 

ovule parenchyma toward the placenta; the chalazal haustorium 

nucleus migrates into this lateral bulge ( Fig. 37 ) to become the 

lateral haustorium nucleus. In some species at least, this lateral 

endosperm haustorium is extremely aggressive. First to be consumed 

are the tapetum cells below the point where the lateral 

haustorium penetrated the tapetum ( Figs. 9, 36, 37 ), followed 

by the parenchyma cells and vascular tissue in the direction of 

the placenta ( Fig. 34 ). In one case, a lateral haustorium had expanded 

all the way into the placenta itself ( Fig. 35 ). Occasionally, 

a second lateral arm through the tapetum is initiated, in the 

opposite direction of the placenta. However, nothing comes of 

this development, possibly because of the lack of a nucleus. 

Extremely aggressive chalazal haustoria may also expand 

exceptionally far in the direction away from the placenta 

( Fig. 35 ). The resulting expansion may be mistaken for a second 

lateral haustorium, but it is located below the bottom of the 

tapetum and has no nucleus. An exceptional case of a lateral 

haustorium growing away from the placenta, i.e., in the opposite 

direction of normal, was observed in N. parvifl ora ( Fig. 8 ). 

This particular ovule had no normal lateral haustorium, i.e., no 

branch growing toward the placenta. The lateral haustorium 

varies greatly in size, from species to species and from ovule to 

ovule within the same species. Very large haustoria were observed 

in ovules of Nemophila menziesii ( Figs. 34, 35 ). Very 

small ones were seen in N. parvifl ora ovules. Apparently, a lateral 

haustorium may not always develop in N. parvifl ora var. 

austinae ( Fig. 11 ). The lateral haustorium is relatively shortlived. 

Its nucleus becomes hypertrophied ( Figs. 34, 35 ) and 

fragmented, then disappears with the lateral haustorium itself, 

while the embryo is still few-celled ( Figs. 38, 39 ). 

Embryo — Embryogeny was studied in detail in Nemophila 

spatulata only. However, individual stages obtained from N. 

parvifl ora ( Figs. 10 – 13 ) and from N. pulchella indicate that the 

embryogeny of these two species, in all probability, is similar to 

that of N. spatulata. 

The embryo does not start developing until eight cells have 

formed in the endosperm proper. At that time, the zygote elongates 

into the micropylar endosperm haustorium, in the form of 

a fi lamentous proembryo, with an apically located nucleus 

( Figs. 8, 9 ). Some time later, the proembryo reaches the endosperm 

proper, forcing its way into it ( Fig. 35 ). The proembryo 

nucleus divides when the endosperm proper consists of 16 

( Fig. 11 ) or more ( Fig. 41 ) cells. The nuclear division is followed 

by transverse wall formation, resulting in a two-celled 

proembryo, with a strikingly small distal (or apical) cell (ca) 

and a long proximal (or basal) cell (cb) ( Figs. 41, 59 ). The proximal 

cell divides more rapidly than the distal. It divides trans- 


569 

versely ( Fig. 10 ), producing a distal (m) and a proximal (ci) 

daughter cell ( Fig. 42 ). Somewhat later, the original distal cell 

(ca) divides transversely ( Fig. 42 ), producing a distal (l) and a 

proximal (l ′ ) daughter cell. Thus, the embryo tetrad (second cell 

generation) consists of four superimposed cells ( Fig. 12 ): l, l ′ , 

m, and ci. Before the next division, the nucleus of ci migrates to 

the middle of its cell, nearly to the outer limit of the endosperm 

proper ( Fig. 43 ). 

Each of the four cells then divides, to produce the eight cells 

of the third cell generation. The two cells l and l ′ divide longitudinally, 

l ′ slightly before l ( Fig. 13 ), producing the four socalled 

quadrant cells ( Cr é t é , 1963 ), arranged in two tiers. The 

most proximal cell (ci) divides transversely, producing the 

daughter cells n (distal) and n ′ (proximal) ( Fig. 13 ). The middle 

cell (m) divides longitudinally, but later than the three other 

cells, producing two juxtaposed cells in the m-tier ( Fig. 44 ). 

Because later cell divisions are not synchronized, I could 

not follow every step in the subsequent development in detail. 

However, in general, the fourth cell generation is characterized 

by the quadrant cells being segmented into “ octant cells ” ( Cr é t é , 

1963 ), normally arranged in two tiers, l and l ′ . The two stages 

closest to the ideal in this material are pictured in Figs. 45 and 

46 . Again, divisions in tier l ′ in this material precede those in 

tier l. The former stage ( Fig. 45 ) shows the two distal quadrant 

cells still undivided (l), while four octant cells have formed in 

tier l , and one of these four is already dividing to produce the 

fi rst “ surface cell ” of this tier. The latter stage ( Fig. 46 ) shows 

four octant cells in tier l, while periclinally orientated, longitudinal 

divisions already have produced eight cells in tier l ′ , four 

inner “ core cells ” and four outer “ surface cells. ” Apparently, in 

the N. spatulata proembryo, divisions in the very apical region 

lag behind divisions in the subapical region to the degree that 

the four “ octant cells ” of the apical region do not appear until 

the four “ octant cells ” of the subapical region have vanished, 

i.e., a true octant is never formed. Within tier m, longitudinal 

divisions produce four juxtaposed cells ( Fig. 46 ). The extreme 

proximal cell n ′ divides transversely into the suspensor cells p ′ 

(proximal) and p (distal). These daughter cells of n ′ are both 

located within the limit of the micropylar haustorium and do 

not divide any further ( Figs. 44 – 47 ). Cell n is the one dividing 

most actively at this stage. It produces the two daughter cells o 

(distal) and o ′ (proximal), through a transverse division ( Fig. 

45 ). The proximal daughter cell (o ′ ) further divides transversely, 

forming the cells r (distal) and r ′ (proximal) ( Fig. 45 ). Finally, 

cell r divides into the superimposed daughter cells s (distal) and 

s ′ (proximal) ( Fig. 46 ). All cells derived from o ′ , that is the 

proximal daughter cell of n, participate in the construction of 

the fi lamentous suspensor. The distal daughter cell of n, i.e., o, 

← 

epidermis and prominent tapetum, and embryo sac with egg apparatus, polar nuclei, and chalazal embryo sac haustorium pushing aside remnants of nucellus 

and antipodal cells. Globules present, but not drawn. 7. Approximately median transverse section from anthetic fl ower, showing cellular composition 

of integument, two-celled endosperm, and embryo sac haustorium. Globules present, but not drawn. 8. As in Fig. 9, but exceptional case of lateral endosperm 

haustorium penetrating in the opposite direction of the placenta. Bar = 100 µ m. 9. Ovule from fl ower showing provascular strand, endosperm 

proper (all eight nuclei indicated), micropylar endosperm haustorium, and chalazal endosperm haustorium with its branch (the lateral endosperm haustorium) 

digesting ovular tissue in the direction of the placenta. 10. N. parvifl ora var. austinae . Two-celled proembryo pushing its way into the endosperm 

proper. Note the large size of cells of outer integumentary epidermis and the short branches produced by the micropylar endosperm haustorium into former 

tapetum. 11. N. parvifl ora var. austinae . Endosperm proper of 16 cells (all nuclei indicated), tapetum reverting to a normal inner integumentary epidermis, 

and chalazal endosperm haustorium with only a small branch toward the provascular strand. 12. Four-celled proembryo (see Results, Embryo ). 13. Sixcelled 

proembryo, cell m undivided, cell l in prophase. Limit of endosperm proper indicated. Abbreviations for all fi gures : c, chalaza; ch, chalazal endosperm 

haustorium; e, embryo/proembryo; em, embryo sac: en, endosperm; ep, endosperm proper; esh, embryo sac haustorium; i, integument; i.e., inner 

epidermis of integument; l, lateral branch of chalazal endosperm haustorium; m, micropyle; mh, micropylar endosperm haustorium; n, nucellus; oe, outer 

epidermis of integument/ovular epidermis; p, placenta; sp, seed pit; t, tapetum; vs, vascular/provascular strand; z, zygote.


Figs. 14 – 26. Longitudinal sections of ovule, nucellus, embryo sac, and endosperm. Figs. 14 – 20, Nemophila pedunculata ; Figs. 21 – 24, 

N. pulchella ; 

Figs. 25, 26, N. heterophylla . 14. Median transverse section from very young bud, showing archesporial cell and early stage of integument. Bar (for all 

fi gures) = 50 µ m. 15. Nucellus enclosing four megaspores, the lowest larger than the others. 16. Integument still not closed above nucellus, lower megaspore 

enlarging, beginning of provascular strand. 17. Two-nucleate embryo sac and early stage of tapetum. 18. Ovule from medium-sized bud. Integument closed 

above nucellus to form micropyle, well-developed tapetum, and eight-nucleate embryo sac penetrating nucellar apex. 19. Mature embryo sac in direct 

contact with tapetum, showing large egg cell, with apical nucleus, smaller synergids, with apical vacuole, polar nuclei, globules presumed to be starch, and 

embryo sac haustorium. 20. Synchronous division in two-celled endosperm, producing four-celled stage. 21. N. pulchella var. fremontii . Median section of 

ovule from very young bud, showing archesporial cell and early stage of integument encircling nucellus. 22. Median transverse section from young bud, 

showing thick integument forming micropyle, nucellus surrounded by tapetum, and lower megaspore enlarging. 23. Nearly median transverse section from 

anthetic fl ower, showing embryo sac in direct contact with tapetum, globules presumed to be starch, embryo sac haustorium, nucellar remnants, and fertilization 

(triple fusion in central cell). 24. Section as in Fig. 23 , but showing zygote and fi rst division in endosperm mother cell. 25. Massive integument 

closed above nucellus to form micropyle, well-developed tapetum, and two-nucleate embryo sac, both nuclei centrally located. 26. Four-nucleate embryo 

sac penetrating nucellar apex. 

may divide early ( Fig. 45 ) or late ( Fig. 46 ) relative to the neighboring 

cells. The division is longitudinal ( Fig. 45 ), producing 

two juxtaposed cells in tier o ( Fig. 47 ). 

The formation of core cells and surface cells in tier l is illustrated 

in Fig. 47 . Two of the four octant cells have divided, one 

by a longitudinally periclinal wall, the other by a transverse 

periclinal wall. The fi rst has resulted in a lateral surface cell and 

a core cell, the second in an apical surface cell and a proximal 

cell. One of the four octant cells is in the process of dividing 

longitudinally. The division is periclinal and will result in an

March 2009] 

other lateral surface cell and another core cell. Because two apical 

surface cells were observed in several proembryos, the last 

octant cell, which still remains undivided in Fig. 47 , most probably 

would have divided transversely, producing the second 

apical surface cell and another proximal cell. Each of the two 

proximal cells, presumably, would have divided by longitudinally 

periclinal walls so that tier l, if not stopped in its development, 

would have come to consist of four core cells, four lateral 

surface cells, and two apical surface cells. The six surface cells 

represent the protoderm initials of tier l. 

The maximum number of suspensor cells eventually might 

surpass 20 ( Figs. 48, 60 ). Longitudinal sections of later stages 

showing the exact number of suspensor cells were impossible 

to obtain. 

Subsequent transverse and longitudinal divisions within tiers 

l, l ′ , and m produce the major part of the globular embryo. The 

derivatives of cell o, also, form part of the globular embryo, 

namely one, later two, tiers of small cells on top of the suspensor 

( Figs. 48, 60 ). Of special importance is the fact that the fi rst 

proximal cell (cb) has given rise not only to the long suspensor 

but, through its derivatives m and o, to a considerable portion of 

the embryo as well. Thus, the embryo development of N. spatulata 

corresponds most closely to the Chenopodiad type, Myosotis 

variation of Johansen (1950, p. 121). 

Embryo of Pholistoma — A correction — In a previous publication 

( Berg, 1985 ), the embryo of P. membranaceum was incorrectly 

described as the Onagrad type as a result of the 

erroneous interpretation of an endosperm nucleus as an embryonic 

tetrad cell nucleus. As a result of this observational error, 

an adjacent three-celled proembryo was misinterpreted as a Tshaped 

embryo tetrad. Additional sectioning of embedded materials 

has now shown beyond doubt that the embryo tetrad of 

P. membranaceum is not T-shaped, but linear, consisting of 

four superimposed cells in a row ( Figs. 49, 50 ). Apparently, the 

embryogeny of this species ( Fig. 51 ) is similar to that of Nemophila 

spatulata : i.e., of the Chenopodiad type, Myosotis 

variation. 

Nemophila seed — After fertilization, the large cells of the 

outer integumentary epidermis continue to enlarge and, for a 

short time, keep pace with the enlargement of the seed. The 

epidermal cells are fortifi ed by rodlike, or hairlike, thickenings 

radiating from the inner wall ( Fig. 62 ). Soon, however, the seed 

outgrows its epidermis in the integumentary region. Here, the 

outer integumentary epidermis becomes broken ( Fig. 39 ) and 

gradually splits into small, disunited cell groups or individual 

cells ( Figs. 40, 61, 63 – 65 ). Only in a limited area around the 

former micropyle does the outer integumentary epidermis remain 

more or less intact until seed maturity ( Fig. 40 ). The integumentary 

parenchyma becomes compressed and is resorbed, 

disappearing before the seed matures. The inner integumentary 

epidermis grows with the seed, due to mitotic divisional activity, 

to become the functional epidermis over most of the seed 

surface ( Figs. 39, 40 ). Its cells are relatively small and isodiametric, 

with a thickened inner wall, thickened radial walls, and 

a much thinner outer tangential wall ( Fig. 64 ). In desiccated 

seeds, the outer wall shrinks into the cell to produce the fi nely 

honeycombed seed surface so characteristic of Nemophila species 

( Fig. 65 ). 

A most unusual thing happens in the developing Nemophila 

seeds. Approximately when the proembryo differentiates into a 

globular embryo proper and a suspensor ( Fig. 39 ), the integument 


571 

and the endosperm drastically change their growth at numerous 

points scattered over the seed surface. At each point, one or more 

cells of the inner integumentary epidermis and the endosperm 

cells on their inside stop growing outward with the surrounding 

cells. The invaginations, which are produced from the “ stopping 

points ” as these are bypassed by the enlarging endosperm, consist 

of thin-walled, embryonic cells derived from the inner integumentary 

epidermis ( Figs. 39, 61, 62 ). An invagination may remain 

narrow ( Fig. 63 ) or become several cell layers wide, and it may 

stay regular or become distorted, all depending upon the amount 

and orientation of cell divisions within it. Finally, cells split from 

each other or degenerate in the middle of the invagination, forming 

a seed pit ( Fig. 64 ). All parenchyma cells within the inner end 

of the young pit disappear. The parenchyma cells lining the pit in 

its outermost part stay intact and eventually acquire thickened inner 

and radial walls similar to those developed in the other cells of 

the seed epidermis. The fully developed seed pits, consequently, 

are lined with seed epidermis in their outer part, but lack this epidermis 

in their inner part ( Fig. 40 ). Because the invagination initials 

developed at irregularly scattered points, the resulting seed 

pits are irregularly scattered over the seed surface ( Fig. 65 ). Because 

an invagination is shaped through the variable divisional 

activity of its embryonic cells, the resulting seed pits may become 

quite different from species to species and from place to place on 

the same seed. Sometimes, the pits are more like wide craters or 

rough excavations on the seed surface. 

Not only the integumentary region of the ovule but also the 

chalazal region develops in an unusual fashion. After the chalazal 

and lateral haustoria have ceased functioning, the remaining 

parenchyma cells continue to enlarge ( Fig. 39 ), as do the seed 

epidermal cells of this region. As a consequence, the chalazal 

end of the ovule grows into a prominent, large-celled seed appendage 

( Figs. 40, 66 ), the so-called cucullus. The cucullus parenchyma 

cells stay relatively thin-walled. The cucullus 

epidermal cells develop somewhat thickened inner and radial 

walls, in addition to weak hairlike thickenings similar to those 

developed in the outer integumentary epidermal cells. Their 

thin outer wall collapses into the cell interior upon desiccation. 

Both kinds of cucullus cells contain globules of fatty acids 

when mature. 

Most of the mature seed is taken up by thick-walled endosperm 

cells ( Fig. 64 ). Because endosperm growth occurs in between the 

simultaneously expanding invaginations ( Figs. 62, 63 ), the endosperm 

becomes pitted. Because the embryo consumes the innermost 

part of the endosperm, nearly out to the region of “ stopping 

points ” (see second paragraph of this section) , the endosperm pits 

extend practically to the seed cavity in mature seeds ( Fig. 40 ). 

The embryo is well developed at seed maturity. Its length is 

approximately two-thirds the length of the seed. It is straight, with 

the two cotyledons somewhat shorter than the hypocotyl – root axis 

( Fig. 40 ). 

DISCUSSION 

Embryology — My results agree with those obtained for Nemophila 

menziesii by Svensson (1925) in several characters: 

The ovule is tenuinucellate and unitegmic; the inner integumentary 

epidermis produces a tapetum; a single archesporial 

cell functions directly as the megaspore mother cell; the embryo 

sac penetrates the nucellar apex and develops according to 

the Polygonum type; the endosperm is cellular; and a micropylar 

and a chalazal endosperm haustorium develop. Of special


Figs. 27 – 35. Longitudinal sections of ovule, embryo sac, endosperm, and young seed in Nemophila maculata ( Figs. 27, 29, 31 – 33 ) and N. menziesii 

( Figs. 28, 30, 34, 35 ). 27. Ovule from young bud with open integument, nucellus, and provascular strand from placenta toward chalaza, showing subepidermal 

megaspore mother cell. Bar (also for Figs. 28 – 33) = 50 µ m. 28. Young embryo sac on top of residual nucellus and in direct contact with tapetum, 

showing young egg apparatus, two polar nuclei, and three antipodal cells. 29. Ovule from anthetic fl ower, showing massive integument closed above embryo 

sac to form micropyle, well-developed tapetum, paired polar nuclei surrounded by globules, and embryo sac haustorium adjacent to remnants of nucellar 

and chalazal cells. 30. Fusion of sperm and egg nuclei and fi rst division of primary endosperm nucleus. 31. Two-celled endosperm. 32. Endosperm 

of four cells in a row. Globules not drawn. 33. Four-celled endosperm, the two middle cells dividing to form the endosperm proper, the nondividing terminal 

cells forming haustoria, globules disappearing. 34. Young seed from withered fl ower, showing 16-celled endosperm proper (all nuclei indicated) and aggressive 

lateral endosperm haustorium, with nucleus, expanding toward vascular strand and placenta. Bar = 100 µ m. 35. Young seed from young capsule, 

showing unicellular proembryo embedded in several-celled endosperm proper, lateral endosperm haustorium extending to placenta, and tapetum reverted

March 2009] 

interest is the fact that I, too, found that an embryo sac haustorium 

is present; that the antipodal cells are extremely shortlived 

and disappear before fertilization; that the tapetum is 

massive and functions as a meristem; and that a lateral endosperm 

haustorium, as a rule, forms from the chalazal endosperm 

haustorium toward the placenta. The lateral endosperm 

haustorium, apparently, may vary in size from species to species 

and from ovule to ovule within the same species. It is particularly 

well developed in N. menziesii , where it may extend 

all the way into the placental tissue (cf. Svensson, 1925, p. 42). 

In N. parvifl ora , on the other hand, the lateral endosperm haustorium 

is quite small or, sometimes, absent. 

Svensson (1925) maintained that the polar nuclei fuse prior 

to fertilization. I could not verify this. On the contrary, triple 

fusion was observed in the central cell. Also, Svensson (1925) 

described and pictured an early endosperm stage of three cells 

in a longitudinal row. Di Fulvio (1987) showed this to be erroneous. 

My results agree with those of DiFulvio. The endosperm 

develops by synchronous divisions directly from being twocelled 

to becoming four-celled, in all species that I studied. 

Embryogeny of N. spatulata was found most closely to follow 

the Chenopodiad type, Myosotis variation of Johansen 

(1950) , agreeing with results obtained for N. menziesii by Cr é t é 

(1947 ; 1963, p.192). 

Seed — Svensson (1925) did not understand seed coat formation 

in Nemophila , and his misunderstandings have produced 

considerable confusion. The tapetum, according to Svensson 

(1925, p. 42), becomes compressed, destroyed, and fi nally, 

completely absorbed, so that no trace remains on the mostly 

naked ( Svensson, 1925, p. 50) seed. This interpretation is completely 

wrong. As demonstrated in the present study, the tapetum 

reverts to a normal inner integumentary epidermis, which 

in time, becomes the minutely reticulate seed coat that characterizes 

this group of Nemophila species (cf. Chuang and Constance, 

1992, p. 261). 

Svensson (1925, p. 49) was somewhat closer to the truth, 

however, when he observed the large-celled outer integumentary 

epidermis to disrupt and gradually become cast off ( “ abgeschabt 

” ). It is clear from my results that the large cells of the 

outer epidermis of the integument soon become disunited 

into widely scattered remnants, as has also been described by 

Chuang and Constance (1992) . It is not unreasonable to assume 

that some of those remnants, fi nally, might come loose and fall 

off individually. However, a gradual casting off of a large piece 

of seed coat, as implied by Svensson ’ s wording, does not occur. 

The formation of seed pits in Nemophila was described by 

Chuang and Constance (1992, p. 261) as resulting from an invagination 

of the integumentary tapetum in certain areas in an 

early stage of seed development. My observations show how 

this invagination occurs. 

The seed appendage of Nemophila has been the source of considerable 

misjudgement and misunderstanding. It was termed a 

cucullus by Brand (1913, p. 21), who justly found previously 

applied terms (arillus, caruncula, calyptra) inappropriate for 

morphological reasons; the Nemophila appendage extends from 

the end of the seed (no arillus) and is attached in the chalazal 

region (no caruncula, no calyptra). Brand (1913) made one good 


573 

and two bad morphological observations. First, he observed that 

the young seed was made of two parts: a darker one with a smallcelled 

epidermis nearest to the placenta and a lighter one covered 

by a large-celled epidermis away from the placenta. The 

darker part was much smaller than the lighter part at the beginning, 

and during seed growth, the darker part enlarged much 

more than the lighter, fi nally to become unquestionably the largest 

of the two seed parts. The small, lighter part became the cucullus. 

This observation is correct. What Brand witnessed was the 

breaking up of the ovular epidermis in the integumentary region 

and the persistence of the ovular epidermis in the chalazal region. 

Second, Brand (1913, p. 21) reported that in some Nemophila 

seeds, those of N. maculata in particular, the cucullus had 

disappeared before the seed reached maturity. This is totally 

wrong, as far as natural processes are concerned. Every single 

Nemophila seed that escapes naturally from its capsule is provided 

with a cucullus. Third, Brand ’ s use of the term “ outer seed 

coat ” implies that he believed the cucullus to be integumentary 

in origin. As demonstrated in the present study, the cucullus 

defi nitely is formed from a part of the ovule that lacks an inner 

integumentary epidermis, i.e., which is not integumentary. 

Brand ’ s conclusions, although wrong, infl uenced the views 

expressed by later investigators. Svensson (1925, pp. 49, 50) 

used the term integument for “ chalaza ” and stated that normally 

( “ gew ö nlich ” ), the entire cucullus “ becomes shed, even though 

it is not uncommon for the cucullus to remain on fully ripe 

seeds. ” Chuang and Constance (1992, p. 263) described the cucullus, 

erroneously, as being derived from the outer epidermis of 

the integument but, correctly, as being attached to the chalaza. 

It seems clear from my results, that it is imperative to an understanding 

of the Nemophila seed to distinguish clearly between 

integument and chalaza. Within the integumentary 

region, there originally exists an inner and an outer integumentary 

epidermis. The outer epidermis becomes large-celled, then 

fragmented and, fi nally, disappears except for small remnants; 

the inner epidermis remains small-celled and becomes the coat 

of the mature seed (cf. Chuang and Constance, 1992, p. 16 ). 

Within the chalazal region, the single ovular epidermis remains 

intact, becoming the epidermis of the cucullus. Parenchymatic 

chalazal cells make up the interior of the cucullus. While the 

integumentary region of the seed produces seed pits, the chalazal 

region produces the cucullus. 

Comparison with other Hydrophylleae — Differences between 

genera of the Hydrophylleae are listed in Table 1 . Eucrypta 

is left out of this comparison because its two species are 

dissimilar in seed morphology and cytology ( Chuang and Constance, 

1992 ), as well as in molecular phylogeny ( Ferguson, 

1999 ). (1) In Nemophila, only one lateral endosperm haustorium 

develops, viz. in the chalaza, while two lateral endosperm 

haustoria develop in Pholistoma , one as a branch from the chalazal 

endosperm haustorium and the other as a branch from the 

micropylar endosperm haustorium ( Svensson, 1925 ; Berg, 

1985, 

fi gs. 50 – 52, 71). DiFulvio (1987) demonstrated that, 

as regards lateral endosperm haustoria, Ellisia is similar to 

Nemophila , while Hydrophyllum is similar to Pholistoma . (2) 

Three genera retain the entire ovular epidermis until seed 

maturity: Hydrophyllum ( Berg, 1984 ; Chuang and Constance, 

← 

to normal epidermis. To the left of the micropylar endosperm haustorium, remnants of collapsed parenchyma cells are indicated among newly formed parenchyma 

cells exterior to regenerated inner epidermis of integument (cf. Fig. 41 ). Bar = 100 µ m.


Figs. 36 – 51. Longitudinal sections of seed, proembryo, and embryo in Nemophila spatulata ( Figs. 36 – 48 ) and of proembryo in Pholistoma racemosum 

( Figs. 49 – 51 ). 36. Young seed from young fruit, showing tapetum, provascular strand, four-celled endosperm proper in two tiers, and initiation of 

lateral endosperm haustorium. Bar = 100 µ m. 37. As in Fig. 36 , but endosperm proper of eight cells in two tiers (all nuclei indicated), and large nucleus is 

moving from chalazal endosperm haustorium into lateral endosperm haustorium. Bar (also for Fig. 38) = 100 µ m. 38. Young seed from medium-sized 

capsule, showing fi liform proembryo embedded in multicellular endosperm proper, isodiametric cells restored from tapetum cells in the inner epidermis of 

the integument, degenerating chalazal and lateral endosperm haustoria, and large-celled outer integumentary epidermis. 39. Half mature seed with fragmenting 

outer integumentary epidermis, showing young seed pits formed by the inner epidermis of the integument, remnants of endosperm haustoria, and 

globular embryo within massive endosperm. Bar = 300 µ m. 40. Mature seed, showing scattered remnants of outer integumentary epidermis, seed epidermis 

formed from inner integumentary epidermis, seed pits through endosperm, large embryo, and chalazal tissue forming cucullus. Bar = 1 mm. 41. Two-celled 

proembryo extending through micropylar endosperm haustorium and into endosperm proper; ca, distal (apical) cell; cb , proximal (basal) cell. Bar (also for 

Figs. 42 – 51 ) = 50 µ m. 42. The proximal cell has divided to form cells ci and m , the distal cell nucleus in metaphase. Outer limit of endosperm proper indi-

March 2009] 

1992 ), Ellisia ( Chuang and Constance, 1992 ), and Pholistoma 

( Berg, 1985 ; Chuang and Constance, 1992 ). Because the ovular 

epidermis covers both the integument and the chalaza, the integumentary 

and the chalazal regions of the seed develop similarly. 

Notably, these three genera have a seed coat of two cell 

layers in the integumentary region, a large-celled outer layer 

from the outer epidermis of the integument and a small-celled 

inner layer from the inner epidermis of the integument. The 

outer of those two layers, with the chalazal epidermis, gives rise 

to the alveolate seed surface characterizing these three genera. 

In the fourth genus, viz. Nemophila, the ovular epidermis stays 

intact only in the chalazal part of the seed, while the smallcelled 

inner epidermis of the integument comes to cover the 

remainder, producing the microreticulate seed surface in this 

genus. (3) Seed pits occur in two genera: Pholistoma and Nemophila 

( Table 1 ). In Pholistoma , the pits are regular in shape 

and arranged in longitudinal rows ( Berg, 1985 ; Chuang and 

Constance, 1992 ), while in Nemophila , as here treated, they are 

irregular both in shape and arrangement. (4) So-called “ giant 

cells ” occur in Pholistoma only. These are extremely large seed 

coat cells, with a conical base that extends far into the seed interior. 

Seed pits in Pholistoma are nothing more than the fortifi 

ed remnants of such “ giant cells ” ( Berg, 1985 ). In Nemophila , 

as here treated, seed pits are formed without the “ help ” of giant 

cells. In Nemophila , seed pits owe their existence to some special 

change in meristematic activity of the inner epidermis of 

the integument. (5) A seed appendage in the form of a chalazal 

cucullus develops only in Nemophila . 

Despite differences in seed structure, Pholistoma is the genus 

most similar to Nemophila . Practically all embryological characters 

studied, including embryogeny according to my reinvestigation 

(see Results, Embryo of Pholistoma ), are identical in the 

two genera. Nemophila deviates embryologically from Pholistoma 

only in the number of lateral endosperm haustoria (Table 

1) and in three minor specialities: (1) the embryo sac haustorium 

is large and expanding transversely in Nemophila ; small 

and expanding longitudinally in Pholistoma ( Svensson, 1925, 

pp. 22, 23; Berg, 1985, fi g. 47). (2) The antipodal cells are extremely 

short-lived in Nemophila , disappearing completely before 

fertilization; while the antipodal cells have a more “ normal ” 

life span in Pholistoma , remnants being recognizable in early 

endosperm stages ( Berg, 1985, fi gs. 46 – 48). (3) At the time of 

fertilization, the tapetum cells in Nemophila are strongly elongated 

radially, producing a moderate amount of integumentary 

parenchyma outward; the tapetum cells in Pholistoma , on the 

other hand, are less elongated radially, but produce a much 

larger amount of integumentary parenchyma outward, the socalled 

central parenchyma ( Berg, 1985, fi gs. 46, 50). 

Taxonomy — Constance (1939a, p. 32) removed two ecucullate 

species from Nemophila to Pholistoma . This transfer is 

strongly supported by the additional information on endosperm 

and seed presented in this paper ( Table 1 ). 


575 

The remainder of Nemophila was treated by Constance (1941, 

p. 345) as a natural genus without further subdivision (see also 

Chuang and Constance, 1992, p. 263). This view has been supported 

by later studies. DiFulvio (1987) showed several of Constance 

’ s Nemophila species to be identical with regard to endosperm 

haustoria. Ferguson (1999, p. 263) described Nemophila as monophyletic 

on the basis of chloroplast gene ndhF studies. My present 

observations of embryology and seed show all Nemophila species 

studied to be similar in all essential characteristics. Nemophila 

seems to be a natural genus also from an embryological point of 

view, when Cave ’ s law (see introduction) is kept in mind. 

However, that Nemophila as conceived by Constance (1941) 

is a natural genus is not necessarily true. DiFulvio (1987) , Ferguson 

(1999) , and the present study were all studies restricted, 

respectively, to fi ve, two, or all of the seven species possessing 

a conspicuous cucullus. The four species with a so-called reduced 

cucullus were not included in any of those studies, and 

these four species differ from the bulk of the Nemophila species 

in both endosperm haustorium development (R. Berg, unpublished 

data) and seed structure ( Constance, 1941 ; Chuang and 

Constance, 1992 ). Probably, the “ reduced cucullus species ” of 

Nemophila will be better placed in a genus of their own. 

Because of similarity in the number of lateral endosperm 

haustoria ( Table 1 ), DiFulvio (1987, p. 31) proclaimed Nemophila 

’ s closest relative to be Ellisia . This view has had little 

support. Constance and Chuang (1982) found that Ellisia fell in 

a different group from Nemophila and Pholistoma , on the criterion 

of pollen exine pattern. The observations on embryology 

presented in this paper for Nemophila are strikingly similar to 

data previously published for Pholistoma ( Berg, 1985 ), indicating 

that the closest relationship is between these two genera. 

However, the taxonomic signifi cance of the embryological similarity 

between Nemophila and Pholistoma cannot be fully 

evaluated until comparable information also becomes available 

for Ellisia , Eucrypta , and Hydrophyllum . Of particular signifi - 

cance is the presence in both genera of such an unusual structure 

as seed pits, which are absent from Ellisia (Table 1). 

Nemophila and Pholistoma were also grouped most closely together 

on the basis of molecular data ( Ferguson 1999, fi g. 3). 

Function and evolution — Two important seed adaptations 

were established during the generic differentiation within the 

tribe Hydrophylleae, viz. seed pits (in Pholistoma and Nemophila 

) and a seed appendage (in Nemophila ). 

I believe that seed pits developed within the Hydrophylleae 

as an adaptation for more rapid water uptake ( Berg, 1985 ; 

Chuang and Constance, 1992 ). It is noteworthy, in this connection, 

that transfer cells facilitating water transport are absent 

from Nemophila seeds ( Diane et al., 2002 ) and that intercellular 

spaces, that might act as water passageways, occur in the seed 

coat of Ellisia ( Chuang and Constance, 1992, fi g. 2). Except for 

Hydrophyllum , the genera in Table 1 are annuals ( Constance 

1939a ), adapted for life during a relatively short season. 

← 

cated. 43. Four-celled proembryo, the distal cell has divided transversely to form the superimposed cells l and l ′ . The nucleus of ci has moved to outer limit 

of endosperm proper. 44. Eight-celled proembryo, l, l ′ , and m (cf. Fig. 13 ) have divided longitudinally to form three two-celled tiers, ci has divided transversely 

into n and n ′ . Note position of cell wall between n and n ′ in relation to limit of endosperm proper! 45. Thirteen-celled proembryo, cell o dividing 

longitudinally (see Results, Embryo ). 46. Twenty-two-celled proembryo, eight cells in tier l ′ consisting of four core cells and four surface cells (see Results, 

Embryo ). 47. Club-shaped proembryo, showing formation of core cells and surface cells in tier l (see Results, Embryo ). 48. Globular embryo on 11-celled 

suspensor, the four tiers l, l ′ , m and o have all become multicellular. 49. Filamentous proembryo made up of four cells in a row: l , l ′ , m , and ci. Outer limit 

of endosperm proper indicated. 50. Four cells as in Fig. 49 , but nucleus of ci has moved to outer limit of endosperm proper. 51. Seventeen-celled proembryo, 

showing four cells in each of tiers l , l ′ , and m , while cell o is still undivided.


Figs. 52 – 64. Light micrographs of longitudinal sections of ovule and seed of Nemophila maculata ( Figs. 55 – 57 ), N. menziesii ( Figs. 53, 54 ), N. parvifl 

ora var. austinae ( Fig. 64 ), N. pedunculata ( Fig. 63 ), N. pulchella var. fremontii ( Figs. 60, 61 ), and N. spatulata ( Figs. 52, 58, 59, 62 ). 52. Anatropous 

ovule, showing single integument closing above small nucellus with linear tetrad of megaspores. Bar = 100 µ m. 53. Nucellus, with linear tetrad, is sur-

March 2009] 

In Pholistoma , the adaptations that result in seed pits can easily 

be viewed as due to a growth change in large seed coat cells, 

similar to those found in Hydrophyllum . “ Something ” stops the 

inner end of every epidermis cell from expanding outward with 

the enlarging ovule. Because new epidermal cells are not added, 

continued growth and enlargement of the ovule result in the 

formation of extremely large, conical epidermal cells, so-called 

“ giant cells. ” Their cell lumens later become seed pits ( Berg, 

1985 ). In Nemophila , the seed pit adaptations do not have such 

a clear relationship to any previously existing seed structure. 

Still, something stops a number of cells scattered within the inner 

epidermis of the integument from continuing their outward 

growth in harmony with their neighboring cells. 

The inner epidermis of the integument in Pholistoma behaves 

in much the same way as the inner epidermis of the integument 

in Nemophila , with one important exception. In Pholistoma , the 

epidermis is clearly prevented from growing outward at certain 

points by the inner tip of a “ giant cell ” ( Berg, 1985, fi gs. 54, 

76). In Nemophila, as here treated, a similar physical restriction 

against outward growth at certain points is lacking. It is diffi cult 

to explain how scattered, irregular seed pits of the Nemophila 


Figs. 65 – 66. Seed of Nemophila spatulata (SEM photos: T. I. Chuang). 65. Seed surface, showing irregularly located seed pits, microreticulate seed 

coat (from inner integumentary epidermis), and scattered exterior cells (remnants of outer integumentary epidermis). Bar = 600 µ m. 66. Cucullus (elaiosome), 

with its large, soft, normally turgid surface cells (from chalazal epidermis) desiccated and collapsed. Bar = 300 µ m. 

577 

type could have arisen directly from an unpitted seed of the type 

found in Hydrophyllum . Much more likely is the establishment 

through an intermediate stage with “ giant cells ” in the integumentary 

region, i.e., Nemophila seed pits and Pholistoma seed 

pits are homologous. With the subsequent phylogenetic disappearance 

of the giant cells, the rigid control of pit arrangement, 

shape, and size disappeared as well. 

The second of the important adaptations is the cucullus. 

Brand (1913) speculated that in earlier times the Nemophila 

seed coat secreted slime, but that now it was functionless and in 

the process of disappearing. He termed the cucullus a functionless, 

“ rudimentary residue of the outer seed coat, ” (p. 21) and 

later found support for this view from Svensson (1925, pp. 49, 

50). The generic monograph by Constance (1941) is infl uenced 

by Brand ’ s terminology and opinions. Constance, too, regarded 

the cucullus as a functionless, “ gradually disappearing remnant 

of an additional seed coat ” (p. 344). However, already at 

the beginning of the 20 th century, Sernander (1906, p. 61) had 

demonstrated that N. menziesii is a myrmecochorous species, 

dispersed by ants because its cucullus functions as an elaiosome. 

In fact, all Nemophila species are myrmecochorous, and 

← 

rounded by tapetum. Bar (also for Fig. 54) = 50 µ m. 54. Two-nucleate embryo sac pushing aside remnants of two intermediate megaspores. Periclinal divisions 

in tapetum have cut off peripheral cells. 55. Four-nucleate embryo sac (one apical nucleus in next section) penetrating nucellar apex. Bar (also for 

Figs. 56 – 60) = 100 µ m. 56. Mature embryo sac enclosed directly by tapetum, showing egg cell, one polar nucleus and embryo sac haustorium. 57. Same 

ovule as in Fig. 56, but next section, showing the two synergids of the egg apparatus and the second polar nucleus. 58. Young endosperm stage, showing 

zygote, synergid remnants, micropylar endosperm haustorium initial (above upper line), two-celled endosperm proper (between lines), and chalazal endosperm 

haustorium (below lower line). 59. Ovular apex, showing two-celled proembryo pushing through micropylar endosperm haustorium and into 

endosperm proper. Tapetum cells reverted into normal inner epidermal cells. 60. Globular embryo on multicelled suspensor within endosperm. Remnants 

of micropylar endosperm haustorium are still visible. 61. Young seed coat, showing groups of large cells derived from fragmented outer integumentary 

epidermis. The small-celled inner integumentary epidermis has formed invaginations into the endosperm. Bar (also for Figs. 62, 63) = 100 µ m. 62. Young 

seed coat, showing outer integumentary epidermis still intact, but larger invaginations have formed into the endosperm. 63. Part of half mature seed, showing 

narrow invagination reaching far into endosperm of thin-walled cells. 64. Part of mature seed, showing seed pit penetrating deep into mature endosperm 

of thick-walled cells. Bar = 200 µ m.


Table 1. Endosperm and seed differences within the Hydrophylleae. 

Trait H E P N 

No. of lateral endosperm haustoria 2 – 3 1 2 1 

Ovular epidermis becomes seed coat + + + – 

Ovular epidermis producing “ giant cells ” – – + – 

Seed pits – – + + 

Seed pits regular and in rows + – 

Seed appendage (cucullus) – – – + 

Habit p/b a a a 

Notes: + = present/yes, - = absent/no, a = annual, b = biennial, p = 

perennial, H = Hydrophyllum , E = Ellisia, P = Pholistoma, N = 

Nemophila (conspicuous cucullus species) 

in all species the cucullus is the ant attractant (R. Berg, personal 

observation; Chuang and Constance, 1992 ). 

How did the cucullus originate? Its surface cells are homologous 

to the chalazal “ giant cells ” of Pholistoma (see Fig. 66 ). 

Obviously, the evolution of a cucullus from cells retaining the 

function of initiating seed pits would meet with strongly confl 

icting selective pressures. A cucullus presupposes the absence 

of pit-producing giant cells from the chalaza. If the Pholistoma 

type of seed were ancestral, the function of pit formation needed 

to be transferred (cf. Corner, 1958 ) from the ovular epidermis 

to the integument, to free the chalaza for cucullus production. If 

the Hydrophyllum type of seed were ancestral, the chalaza 

would be free for cucullus production, but without giant cells, 

how could seed pits evolve? Again, an answer might rest with 

the “ reduced cucullus ” species of Nemophila . 

According to Chuang and Constance (1992, fi gs. 13 – 20), the 

“ reduced cucullus ” species of Nemophila have a cucullus combined 

with a seed surface much like the one in Pholistoma , and 

they do also possess “ giant cells ” (R. Berg, unpublished data). 

A study of the “ reduced cucullus ” species of Nemophila , therefore, 

might possibly show how seed pits and a cucullus could 

evolve side by side within the Hydrophylleae. 

A third adaptation in the evolutionary history of tribe Hydrophylleae 

is the development of lateral endosperm haustoria, 

which function in the transfer of materials from ovular tissues to 

the endosperm. It is confusing that one of the unpitted, presumably 

more primitive genera, namely Hydrophyllum , and one of 

the pitted and presumably more advanced genera, Pholistoma , 

both develop two lateral haustoria, in some species of Hydrophyllum 

even three, according to DiFulvio (1987) , while the 

other unpitted genus, Ellisia , as well as the other pitted genus, 

Nemophila , both develop only one lateral (in both genera from 

the chalazal haustorium). As described here earlier, a tendency 

to form additional lateral haustoria was observed in Nemophila, 

but this tendency was ontogenetically suppressed. Possibly, the 

haustorium branch has evolved from several to one and done so 

more than once (cf. DiFulvio, 1987 , fi gs. 10, 11). 

Concluding remarks — This study makes Nemophila one of 

the best-known genera of angiosperms with regard to embryology 

in a broad sense. Nemophila is characterized by two unusual 

features, viz. a seed cucullus, which acts as an elaiosome 

producing seed dispersal by ants, and seed pits, which probably 

facilitate water uptake and germination. This study provides a 

detailed description of the ontogeny and anatomy of these two 

unusual characteristics, thereby correcting and clarifying earlier 

results by other authors. The new information on embryology 

and seed anatomy strongly supports Constance ’ s removal 

of two Nemophila species to Pholistoma . Also, the new evi- 

dence clearly favors the view that the investigated species, i.e., 

the “ conspicuous cucullus species ” of Constance, form a natural/monophyletic 

genus and, furthermore, that the closest relative 

of Nemophila is Pholistoma . 

LITERATURE CITED 

APG II [Angiosperm Phylogeny Group II ]. 2003 . An update of the 

Angiosperm Phylogeny Group classifi cation for the orders and families 

of fl owering plants: APG II. Botanical Journal of the Linnean 

Society 141: 399 – 436. 

Berg , R. Y. 1984 . Fr ø anatomi, systematikk og evolusjon innen honningurtfamilien 

(Hydrophyllaceae). Det Norske Videnskaps-Akademi, 

Å rbok 1982: 9 – 13. 

Berg , R. Y. 1985 . Gynoecium and development of embryo sac, endosperm, 

and seed in Pholistoma (Hydrophyllaceae) relative to taxonomy. 

American Journal of Botany 72 : 1775 – 1787 . 

Brand , A. 1913 . Hydrophyllaceae. In A. Engler [ed.], Das Pfl anzenreich, 

vol. 4, no. 251, 171 – 220. Engelmann, Leipzig, Germany. 

Cave , M. S. 1953 . Cytology and embryology in the delimitation of genera. 

Chronica Botanica 14 : 140 – 153 . 

Chuang , T. I. , and L. Constance . 1992 . Seeds and systematics in 

Hydrophyllaceae: Tribe Hydrophylleae. American Journal of Botany 

79 : 257 – 264 . 

Constance , L. 1939a . The genera of the tribe Hydrophylleae of the 

Hydrophyllaceae. Madro ñ o 5 : 28 – 33 . 

Constance , L. 1939b . The genus Pholistoma Lilja. Bulletin of the Torrey 

Botanical Club 66 : 341 – 352 . 

Constance , L. 1941 . The genus Nemophila Nutt. University of California 

Publications in Botany 19 : 341 – 398 . 

Constance , L. , and T. I. Chuang . 1982 . SEM survey of pollen morphology 

and classifi cation in Hydrophyllaceae (waterleaf family). American 

Journal of Botany 69 : 40 – 53 . 

Corner , E. J. H. 1958 . Transference of function. Journal of the Linnean 

Society, London , Botany 56 : 33 – 40 . 

Cr é t é , P. 1947 . Embryog é nie des Hydrophyllac é es. D é veloppement de 

l ’ embryon chez le Nemophila insignis Benth. Comptes Rendus de 

l ’ Acad é mie des Sciences, Paris 224: 749 – 751. 

Cr é t é , P. 1963 . Embryo. In P. Maheshwari [ed.], Recent advances in the 

embryology of angiosperms, 171 – 220. Catholic Press, Ranchi, India. 

Diane , N. , H. H. Hilger , and M. Gottschling . 2002 . Transfer cells in the 

seeds of Boraginales. Botanical Journal of the Linnean Society 140 : 

155 – 164 . 

Di Fulvio , T. E. 1987 . La endospermogenesis en Hydrophylleae 

(Hydrophyllaceae) con relaci ó n a la taxonom í a. Kurtziana 19 : 13 – 34 . 

Di Fulvio De Basso , T. E. 1990 . Endospermogenesis y taxonom í a de la 

familia Hydrophyllaceae y su relaci ó n con las demas Gamopetalas. 

Monograf í as de la Academia Nacional de Ciencias Exactas , F í sicas y 

Naturales, Buenos Aires 5 : 73 – 82 . 

Ferguson , D. M. 1999 . Phylogenetic analysis and relationships in 

Hydrophyllaceae based on ndhF sequence data. Systematic Botany 

23 : 253 – 268 . 

Hofmeister , W. 1858 . Neuere Beobachtungen ü ber Embryobildung der 

Phanerogamen. Jahrb ü cher f ü r Wissenschaftliche Botanik 1 : 82 – 188 . 

Johansen , D. A. 1940. Plant microtechnique. McGraw-Hill, New York, 

New York, USA. 

Johansen , D. A. 1950 . Plant embryology. Chronica Botanica, Waltham, 

Massachusetts, USA. 

J ö nsson , B. 1881 . Om embryos ä ckens utveckling hos Angiospermerna. 

Lunds Universitets Å rsskrift 16 : 1 – 86 . 

Schnarf , K. 1931 . Vergleichende Embryologie der Angiospermen. 

Gebr ü der Borntraeger, Berlin, Germany. 

Sernander , R. 1906 . Entwurf einer Monographie der europ ä ischen 

Myrmekochoren. Kungliga Svenska Vetenskapsakademiens 

Handlinger 41 : 1 – 410 . 

Svensson , H. G. 1925 . Zur Embryologie der Hydrophyllaceen, 

Borraginaceen und Heliotropiaceen mit besonderer R ü cksicht auf die 

Endospermbildung. Uppsala Universitets Å rsskrift, Matematik och 

Naturvetenskap 1925(2): 1 – 176.

March 2009] 


Appendix 1. List of fi xed material from wild populations in Californian counties, of seed for cultivation, and of herbarium material for SEM. Voucher specimens are 

deposited as indicated: (O) Botanical Department, Natural History Museum, University of Oslo, Oslo, Norway; (DAV) John M. Tucker Herbarium, University 

of California, Davis, California, USA; and (MO) Herbarium, Missouri Botanical Garden, St. Louis, Missouri, USA. 

Taxon — Locality, Date of fi xation/collection, (Herbarium). 

Nemophila maculata Lindley — Placer: along Auburn-Folsom Road, ca. 

13 km S of Auburn, 15 April 1954 (O) and, seeds only, 22 May 1955; 

— Eldorado: between Alder Creek Campground and Kyburz, 6 May 

1955 (O). 

N. menziesii Hook. & Arn. — Napa: Putah Creek Canyon, 3.2 km S of 

Monticello, 8 April 1954 (O); — Placer: along Auburn-Folsom Road, ca. 

13 km S of Auburn, 15 April 1954 (O); — Fresno: Highway 180 at ca 800 

m, above Clingan ’ s Junction, 24 April 1955 (O). 

N. parvifl ora Benth. — Marin: Woodacre, 16 April 1955 (O). — Marin: Alpine 

Lake-Mt. Tamalpais Road, 3 June 1955 (O) and, seeds only, 2 July 1955. 

N. parviflora var. austinae (Eastw.) Brand — Modoc: Cedar Pass 

Campground, W of Cedarville, 21 June 1955 (O) and, including seeds, 

27 July 1955. 

579 

N. pedunculata Benth. — Modoc: Ceder Pass Campground, W of Cedarville, 

21 June 1955 (O) and 27 July 1955. 

N. pulchella Eastw. — Fresno: Highway 180 at ca. 800 m, above Clingan ’ s 

Junction, 24 April 1955 (O); — Fresno: Highway 180 at ca 1150 m, below 

Snowline Lodge, including seeds, 6 July 1955. 

N. pulchella var. fremontii (Elmer) Constance — Kern: between Miracle 

(Hobo) Hot Springs and mouth of Kern Canyon, 26 April 1955 (O) and 14 

April 1963 (DAV, O). 

N. spatulata Coville — Eldorado: between Camp Richardson and Fallen Leaf 

Lake, 6 May 1955 (O) and 21 July 1955 (O); Eldorado: meadow at ca. 

2400 m, just W of Luther Pass on Highway 89, including seeds, 1 August 

1955 (O); — Butte: between Appleton and Coutolenc, Heller 13177 (MO), 

herbarium materials for SEM.

Rolf Y. Berg 2 - American Journal of Botany

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?