BIOSYSTEMATIC ANALYSIS OF ADIANTUM VIVESII PROCTOR

(POLYPODIACEAE: ADIANTOIDEAE), A RARE FERN ENDEMIC TO

NORTHWESTERN PUERTO RICO

by
Marian Tamari Sepúlveda-Orengo

A thesis submitted in partial fulfillment

of the requirements for the degree of

MASTER IN SCIENCE
in
BIOLOGY

UNIVERSITY OF PUERTO RICO

MAYAGÜEZ CAMPUS
2000
Approved by::

_________________________________ _______________
Inés Sastre-De Jesús, PhD Date
Member, Graduate Committee

_________________________________ _______________
Gary J. Breckon, PhD Date
Member, Graduate Committee

_________________________________ _______________
Duane A. Kolterman, PhD Date
President, Graduate Committee

_________________________________ _______________
Sally González Miranda, PhD Date
Representative of Graduate Studies

__________________________________ _______________
Carlos A. Muñoz, PhD Date
Associate Director and
Coordinator for Graduate Studies in Biology

__________________________________ _______________
Jaime Seguel, PhD Date
Director of Graduate Studies
 Abstract

Adiantum vivesii Proctor is a rare fern endemic to the northwestern part of

Puerto Rico, known only from its type locality on limestone substrate on privately

owned land in the Municipality of Quebradillas. The distribution, abundance,

morphology, and cytology of the species were studied to clarify its status as a distinct

species or of hybrid or polyploid origen. The population of A. vivesii occupies a total

area of 21 m x 10 m; eight sympatric species of Adiantum are found in the immediate

area. Following several deep excavations it was concluded that the entire population

of A. vivesii is one individual with proliferation by rhizomes. Morphological

measurements and light and scanning microscopy were used to describe some parts of

A. vivesii and A. tetraphyllum. Morphometric studies were conducted for 22 characters

of the species and the possible parent, Adiantum tetraphyllum. Greater variability was

obtained within the A. tetraphyllum population, and statistically significant differences

between the two species were observed for 17 characteristics. Meiotic studies were

inconclusive, but no evident difference in chromosome number was observed between

the two species. The spores of A. tetraphyllum showed normal appearance and were

rarely abortive, while those of A. vivesii showed greater variability in size and were

almost always abortive, suggesting that A. vivesii is a sterile hybrid.

ii
 Resumen

Adiantum vivesii Proctor es un helecho raro y endémico de la parte noroeste de

Puerto Rico, sólo conocido de su localidad tipo en suelos cársicos en terrenos privados

del Municipio de Quebradillas. La distribución, abundancia, morfología y citología de

la especie fueron estudiados para aclarar el estado de la especie como distinta, un

híbrido o de origen poliploide. La población de A. vivesii ocupa un área total de 21 m

x 10 m; se encuentran ocho especies simpátricas de Adiantum en el área inmediata.

Después de algunas escavaciones profundas se concluyó que la población de A. vivesii

consta de un solo individuo con proliferaciones del rizoma. Medidas morfológicas y

microscopía de luz y rastreo fueron utilizadas para describir algunas partes de A. vivesii

y A. tetraphyllum. Estudios morfométricos fueron conducidos para 22 caracteres de la

especie y del posible progenitor Adiantum tetraphyllum. Se obtuvo mayor variabilidad

dentro de la población de A. tetraphyllum; 17 características fueron encontradas con

diferencias estadísticamente significativas entre las especies. Estudios meióticos

fueron inconclusos, pero no se observó una diferencia evidente en el número

cromosómico entre las dos especies. Las esporas de A. tetraphyllum mostraron una

apariencia normal y raramente estuvieron abortivos, mientras que las de Adiantum

vivesii mostraron mayor variabilidad en el tamaño y casi siempre estuvieron abortivas,

lo cual sugiere que A. vivesii es un híbrido estéril.

iii
 Dedication

I dedicate this work first of all to God. To my parents Neftalí and Marina, my

sister Marian Talimar and my brother Neftalí for all their endless love and support.

Especially in memory of my beloved grandmother María Elena Collazo Rivera (1915-

2000).

iv
 Acknowledgments

To God, because with Him nothing shall be impossible. To my parents, Marina

and Neftalí, my brother Neftalí (Tito), for their financial and emotional support in

these years: I love you all. Especially, to my sister Marian Talimar (Cachin), without

your help this seemed impossible, thanks. To Dr. Duane Kolterman, thanks for giving

me an opportunity to demonstrate what I can do and to accept being my thesis advisor.

To Dr. Gary Breckon and Dr. Inés Sastre thanks for being an important part of this

thesis, believing in me, and helping my dreamed came true. To Dr. Carlos Alberto

Muñoz, thanks for your advice in all these years, your patience and sincere friendship.

To Dr. Dave Conant and Dr. Christopher Haufler, thanks for your advice and help.

Thanks for the field assistance of Mr. Miguel (Papo) Vives, Mr. William Estremera and

Augusto Carvajal; it is not an easy place to go. To José (Tito) Almodóvar, thanks for

your help with the scanning microscope. To Antonio Seda, thanks for lending me your

computer and your time to make me feel better in the bad times. To Felipe Pagán, you

brougth me peace when I needed it and a sincere friendship. To Tiendas La Gloria,

Inc. and all the employees, especially my partners in the Computer Center; Annette,

Desiree, Juan, Yoly, Yalettsy, Freddy, Elizabeth and Charianne, thanks for giving me

the opportunity of being part of your family and your friendship. To my friends;

Abeliz, Carmen, Jeanine, Lilliam, Katerine, Denisse, Jason, Oscar, Hilcia, Gloria,

Heidi, José Arnaldo, Vivian, and Glorivee all of you shared with me the love for

botany. To Nelly Doris Medina, my best roommate, for all the patience you have
v
with me all these years. To Angelita Leyva, thanks for all your love and friendship. To

María Cruz, thanks for your company last summer and your friendship. To a special

person that brought his support for all these years and take care of me and my partners

in the nights, when we worked for the laboratories or for the classes, Don Luis, thanks

for all your helped in these years, you are an important part of the Biology Department.

Thanks you all,

Marian

vi
 Table of contents

List of tables…..………………….……………………………….……… viii

List of figures………………….………………………………….…….… ix

Introduction………………………………………………………………. 1

Literature Review…………………………………………………….….. 6

General floristic treatments………………………………………….…. 6

Ecological studies…………………………………………….…….….. 7

Morphological studies………………………………………………….. 8

Cytological studies…………………………………………………..…. 11

Hybrid studies………………………………………………………….. 13

Materials and Methods ……....………………………………….……… 14

Study area………………………………………………………………. 14

Distribution and abundance……………………………………………. 15

Morphometric analysis…………………………………………………. 16

Chromosome counts……………………………………………………. 16

Light microscopy……………………………………………………….. 17

Scanning electron microscopy (SEM)…………………………………. 18

Results……………………………………………………………………. 21

Distribution and abundance……………………………………………. 21

vii
 Morphometric analysis…………………………………………………. 22

Chromosome counts……………………………………………………. 22

Light microscopy…………………………………………………..…… 23

Scanning electron microscopy (SEM)…………………………………. 24

Discussion ….……………………………………………….…………….. 25

Conclusions …………………………………………………….…………. 31

Recommendations ……………………………………………………..…. 32

Literature Cited …..…………………………………….……………….. 33

Tables ………………………………………………………….………….. 38

Figures ……………………………………………………………………. 49

viii
 List of tables

Table 1. List of features included in morphometric analyses ……………………. 39

Table 2. Species associated with Adiantum vivesii, according to Miguel A.Vives .. 40

Table 3. Comparison of some characteristics of sympatric Adiantum species, based on

Proctor (1989) …....................……..………………………………...............46

Table 4. Results of a morphometric analysis of Adiantum vivesii and A. tetraphyllum

based on 21 vegetative characters and one spore character .................……..47

ix
 List of figures

Figure 1. Topographic map of Barrio San Antonio, Quebradillas……..……....…. 50

Figure 2. Leaf features included in morphometric analysis………………...…….. 51

Figure 3A. Rhizome connections between the apparent individuals of Adiantum

vivesii …………..…..…………….................……………………..….…............ 52

Figure 3B. Rhizome of Adiantum vivesii about half a meter long …….…..…...... 52

Figure 3C. New plant of Adiantum vivesii, grown from a rhizome collected in March
1999 …………………..…………………………………………........................ 52

Figure 3D. New plant of Adiantum vivesii, grown from a rhizome collected in March
2000 ……………………….………………………….…………........................ 52

Figure 4. Dotplot of petiole length in Adiantum vivesii and Adiantum tetraphyllum

……………….………………………………….....................…………............. 53

Figure 5. Dotplot of blade length in Adiantum vivesii and Adiantum tetraphyllum

………………………...…………………......................…………..……............. 54

Figure 6. Dotplot of blade width in Adiantum vivesii and Adiantum tetraphyllum

………….…………………………………………………................................... 55

Figure 7. Dotplot of blade length/width in Adiantum vivesii and Adiantum

tetraphyllum …………….……………………………….………………............ 56

Figure 8. Dotplot of number of lateral pinnae in Adiantum vivesii and Adiantum

tetraphyllum ………..…………………………………………............................ 57

Figure 9. Dotplot of mean length of lateral pinnae in Adiantum vivesii and Adiantum
tetraphyllum ……………….…………………………………............................. 58

Figure 10. Dotplot of mean width of lateral pinnae in Adiantum vivesii and Adiantum
tetraphyllum ……………...…………….……...………..……............................. 59

Figure 11. Dotplot of mean length/width of lateral pinnae in Adiantum vivesii and
Adiantum tetraphyllum ….....……………….………….................................….. 60

x
Figure 12 Dotplot of distance between first and second lateral pinnae in Adiantum
vivesii and Adiantum tetraphyllum ………….……..…..….............................…... 61

Figure 13 Dotplot of number of pinnules on lateral pinnae in Adiantum vivesii

and Adiantumtetraphyllum …………………………………...…………..........… 62

Figure 14 Dotplot of mean length of longest pinnule on lateral pinnae in Adiantum

vivesii and Adiantum tetraphyllum ………………………...............................….. 63

Figure 15 Dotplot of mean width of longest pinnule on lateral pinnae in Adiantum

vivesii and Adiantum tetraphyllum ……………….….....….........................….…. 64

Figure 16 Dotplot of mean length/width of longest pinnule in lateral pinnae in

Adiantum vivesii and Adiantum tetraphyllum ………..............................….…….. 65

Figure 17 Dotplot of length of terminal pinnae in Adiantum vivesii and Adiantum

tetraphyllum …….……………..............……..………………................…..….… 66

Figure 18 Dotplot of width of terminal pinna in Adiantum vivesii and Adiantum

tetraphyllum …………….……………..……..…..............................……….…… 67

Figure 19 Dotplot of length/width of terminal pinnae in Adiantum vivesii and

Adiantum tetraphyllum …………….………………………...............…..………. 68

Figure 20 Dotplot of petiolule length in Adiantum vivesii and Adiantum

tetraphyllum ………..…..…….………………….................…..…………………69

Figure 21 Dotplot of number of pinnules on terminal pinnae in Adiantum vivesii and

Adiantum tetraphyllum …..…….…….................................….……………....…. 70

Figure 22 Dotplot of length of largest pinnule on terminal pinnae in Adiantum vivesii

and Adiantum tetraphyllum …..……….…………............................…………… 71

Figure 23 Dotplot of width of largest pinnule on terminal pinnae in Adiantum vivesii

and Adiantum tetraphyllum …..…..…….…………….….............................…… 72

Figure 24 Dotplot of length/width of largest pinnule on terminal pinnae in Adiantum

vivesii and Adiantum tetraphyllum ……………………..............................…….. 73

Figure 25 Dotplot of spore size (mm) in Adiantum vivesii and Adiantum

tetraphyllum ………..………………………….…............………………..……. 74

xi
Figure 26. Stages of meiosis in sporogenous tissue in Adiantum vivesii ……….... 75

Figure 27A. Adiantum vivesii pinnule with sori …………………...….………..... 76

Figure 27B. Adiantum vivesii mature sporangia ……………...…………….…..... 76

Figure 27C. Mature sporangia of Adiantum vivesii ...…………………………..... 76

Figure 27D. Mature opened sporangia of Adiantum tetraphyllum .…………….... 76

Figure 28A. Contraction of annulus and lip cells of Adiantum vivesii ...……….... 77

Figure 28B. Abortive spores of Adiantum vivesii (20x) .....…………………….... 77

Figure 28C. Close-up of abortive spores of Adiantum vivesii (40x) ….…..……... 77

Figure 28D. Adiantum tetraphyllum normal spores (20x) ……………………..... 77

Figure 28E. One abortive spore in Adiantum tetraphyllum …………………....... 77

Figure 29A. Scanning photograph of rhizome in Adiantum vivesii …………....... 78

Figure 29B. Scanning photograph of immature sporangia in Adiantum vivesii ..... 78

Figure 29C. Scanning photograph of mature sporangia in Adiantum vivesii ......... 78

Figure 29D. Close-up of a scanning photograph of mature sporangia in Adiantum

vivesii ...………..…………….………………….……………............................. 78

Figure 30A. Scanning photograph of spores of Adiantum vivesii .....…………...... 79

Figure 30B. Scanning photograph of Adiantum tetraphyllum spores ...………...... 79

Figure 30C. Close-up of a scanning photograph of Adiantum tetraphyllum spores

………………..………….…………………………………...……...................... 79

xii
 Introduction

Pteridologists have thought that many of the mysteries involving speciation

and species concepts in pteridophytes could be addressed only by additional and more

intense studies of tropical groups (Haufler, 1996). In fact, evidence indicates that it is

in such systems that the most rapid speciation is occurring. Investigations of tropical

pteridophytes have suggested that speciation in these plants may differ qualitatively

from that in temperate groups (Haufler, 1996).

Probably all named fertile pteridophyte species are morphological species;

even when other criteria are employed, morphological discrimination of taxonomic

units has been an important component of naming new species. No matter how

sophisticated pteridologists become in applying molecular techniques, the practical

need to identify plants in the field will demand that they provide morphological

criteria for differentiating taxa. Evidence from past research and from new analyses

indicates that most pteridophyte species are reproductively isolated units. Fewer data

are known on whether populations at opposite ends of the ranges of widespread

species retain the capacity to interbreed. Available data have demonstrated that

hybrids between congeners are sterile; there are only few cases of fertile F1s.

Pteridologists know far too little about tropical groups and, through expanded

application of biosystematic methods in studying such groups, they may discover that

1
 “biological” species are less numerous than morphological species.

Nevertheless, the evidence to date supports the sterility of hybrids (Haufler, 1996).

Analyses of gametophyte reproductive biology are necessary to develop a

complete picture of the biology of fern species as well as to characterize evolutionary

tendencies. Analyses of chromosomal behavior at meiosis have been especially

important in characterizing suspected hybrid individuals. Blasdell (1963) and Moran

(1981) noted the value of spore features for identifying hybrids and determining the

ploidy levels of specimens. It may be that tropical species have more specific

requirements for successful spore germination and gametophyte maturation than those

in temperate zones, and that gametophytes play a more important role in the

speciation process in the tropics (Haufler, 1996).

The genus Adiantum L. is a large and diverse group of ferns in the Old and

New World tropics. Characterized by submarginal sori borne abaxially on recurved

lobes of the leaf margin (false indusium), it is one of the most clearly circumscribed

genera in the Order Filicales or Polypodiales (Bower, 1928; Tryon, 1964, cited in Paris

and Windham, 1988). Proctor puts this genus in the family Polypodiaceae, subfamily

Adiantoideae.

Adiantum is a large pantropical genus of perhaps nearly 200 species, most

numerous in South America, with a few occurring in or extending into temperate

regions. In English, many of them are commonly called “maidenhair ferns.” The

2
 generic name Adiantum is of Greek origin and means unwetted, referring to

the rain-shedding texture of the foliage in many species (Proctor, 1989). The cytology

of the genus is fairly consistent, with haploid chromosome numbers being n=29, 30, or

multiples of these. The fossil record of this family is only known definitively from the

Eocene.

In Puerto Rico we have a rich and diverse pteridophyte flora. Many of them

are well characterized, but in the case of Adiantum vivesii Proctor, described by Dr.

George R. Proctor in 1985 from specimens collected with Miguel Vives and William

Estremera in the San Antonio Ward in the municipality of Quebradillas (Proctor,

1989), we do not know whether it is a distinct species or of hybrid or polyploid origin.

This fern is known only from the type locality on limestone substrate in Quebradillas,

in northwestern Puerto Rico, where it occurs on privately owned lands. It is endemic

to Puerto Rico and has been Federally listed as an endangered species since June 9,

1993 (U.S. Fish and Wildlife Service, 1994).

In his Latin diagnosis of A. vivesii, Proctor (1989) distinguishes the species

from A tetraphyllum Humb. & Bonpl. ex Willd. based on its fewer pinnae, fewer

pinnules on the lateral pinnae, and submedial costa on the pinnules. He believes that

A. tetraphyllum represents the closest relative of A. vivesii (G. R. Proctor, pers.

comm.).

3
 Many endangered plant species exist in unique ecological niches, such as

those found in bogs, on special soil types, on isolated islands, in mountain ranges, or

in valleys. They are rare because they are adapted precisely to their present location

and will survive only if their habitat is preserved. Certain types of plants are highly

prized by plant fanciers. These particularly include cacti, carnivorous plants, cycads,

orchids, ferns, palms, and bromeliads. The professional botanist who is collecting

uncommon plants for proper study and understanding needs to exercise caution

regarding how many specimens are collected and how the plants are cared for, as well

as adhering to laws governing plant collecting (Woodland, 1997).

Destruction or modification of its habitat may be the most significant factor

potentially affecting the abundance of this endemic fern. Clearing or development of

the area would result in the elimination of the only known population. Also, this

species could be an attractive item for collectors. The extreme rarity of this species

makes the loss of individual plants even more critical. At the same time, the plant is

rhizomatous and it is possible that the entire population consists of only one or a few

individuals. The accessibility to the area is difficult; the owner has only allowed the

entrance of people who are doing research on the fern. He did not know the exact

place to find the population, and it would be very difficult to find it without a guide.

Also it is dangerous to go to the area alone because of the cliffs and only people with

4
 experience in the field can get there without problems. Therefore, there is

essentially no human impact in the area.

The objectives of this project were the following:

1) to determine the distribution and abundance of Adiantum vivesii,

2) to conduct a morphometric analysis of selected leaves of Adiantum

vivesii and Adiantum tetraphyllum Humb. & Boupl. ex Willd.

3) to count the chromosomes of A. vivesii,

4) to document selected parts of A. vivesii and, in some cases, A.

tetraphyllum using the dissecting microscope and the scanning electron

microscope (SEM).

5) to determine whether A. vivesii makes well formed, viable spores.

The principal goal of this project was to clarify the status of Adiantum vivesii

as a distinct species or one of hybrid or polyploid origin. In addition, the present study

represents the implementation of some tasks specified in the recovery plan for the

species (U.S. Fish and Wildlife Service, 1994). Some of those tasks are: to monitor

the population, to study the reproductive biology and ecology of the fern, to assess the

periodicity of spore production and dispersion, and to assess spore viability and

germination requirements.

5
 Literature Review

General floristic treatments

In 1960, Scamman revised the maidenhair ferns (Adiantum) of Costa Rica; in

his article he includes distribution data, a key, and illustrations of the 28 species. A

key to all the Adiantum species of Central America was done by Seymour (1975).

Palacios-Ríos and Riba (1984) made a key to the 20 species of Adiantum of Veracruz,

including synonymy and brief descriptions. Pteridophytes were included in Britton

and Wilson´s flora of Puerto Rico and Virgin Islands (Maxon, 1926), and ferns and

fern allies of the same region were recently revised by Proctor (1989). He recognizes

408 pteridophyte taxa, among which Adiantum includes 15 native species (one with

two varieties) and two additional cultivated taxa. He describes each species, including

its general and local distribution, habitat, and synonymy. Kepler (1975) published an

illustrated guide to the common ferns of the Luquillo Forest. In 1992, Rodríguez

published a list of the ferns in the Maricao Forest, and Cedeño-Maldonado (1997) also

included pteridophytes in his flora of the Río Maricao drainage. In volume one of

Flora Mesoamericana the genus Adiantum was treated in three seminatural groups

including a total of 36 species. A natural subgeneric classification awaits a worldwide

study of the genus (Moran et al., 1995).

6
 Many fern species complexes have been studied. For example, the Adiantum

pedatum complex has been studied since 1949 by R. Wylie, as well as by Wagner and

Boydston (1978), Cody (1983), Paris and Windham (1988), and Rabe and Haufler

(1992). Other species complexes that have been studied include the Cystopteris

tennesseensis complex (Haufler et al., 1990; Paler and Barrington, 1995) and the

Polypodium vulgare complex (Haufler et al., 1995a, 1995b).

De la Cruz (1998) worked with the fern Cyathea brooksii Maxon

(Cyatheaceae), apparently restricted in Puerto Rico to serpentine-derived soils within

the Maricao Commonwealth Forest. Her study included the distribution, relative

abundance, phenology, population ecology, reproductive biology, propagation, and

genetic variability of the species.

Ecological studies

As regards studies of Adiantum, in addition to those of the A. pedatum

complex mentioned above, Sharma and Harsh (1992) studied three species of the

genus from Rajasthan, in northwestern India. They found that, in addition to

ecomorphological variation, the plants also show differences in anatomy and sex

expression in the gametophyte.

The distribution patterns of Adiantum species at twelve noninundated rain

forest sites in Amazonian Peru and Ecuador were studied by Tuomisto-Hanna et al.

7
 (1998). The occurrence and abundance of the species varied among sites with

regard to soil texture and the concentration of exchangeable bases and within sites

with regard to topography. They concluded that ecological differences among the

species clearly explained many of the differences in their distribution patterns at local

and regional scales and may be important for the understanding of such patterns at

biogeographical scales.

Prado and Palacios-Ríos (1998) discussed the taxonomy and

geographical distribution of Adiantum trapeziforme L. and A. pentadactylon Langsd.

& Fisch to establish the distinctness between them. They found that the two species

differ morphologically and that confusion between them has resulted from

misidentification. They presented a new key to distinguish the species. Also A.

pentadactylon is an endemic and locally common species in southeastern Brazil while,

in contrast, A. trapeziforme is a widespread and locally common species from

portions of Mexico throughout Mesoamerica, Cuba, Jamaica, the Lesser Antilles to

Trinidad, and possibly Venezuela.

Morphological studies

Palmer (1998) used morphological characters to distinguish Cibotium x

heleniae hyb. nov. from its parents Cibotium chamissoi and Cibotium menziesii. The

characters that he used were: hairs color and distribution on the stipe, and the depth to

8
 costa and angle of the sinuses. He found that plants of C. x heleniae are

generally intermediate between its known parents. The spores of the hybrid vary in

morphology and their fertility also depends on where they were collected. Only in

one of the study sites were normal spores collected that germinated and formed

gametophytes, but the reason for this variability is not known.

Morphological comparison of parents and potential hybrids and

chemosystematic methods are well tested techniques that have been successfully

employed to demonstrate hybridity in ferns. While the morphological features of a

hybrid usually tend to be intermediate, chemical components often are inherited

additively from each parent. Within the ferns, chemical data based on flavonoids have

been useful in elucidating the parental origin of hybrids in Osmunda and Asplenium,

and in determining the alloploid and autoploid origin of two tetraploid chemotypes of

Pityrogramma triangularis (Conant and Cooper-Driver, 1980).

A morphometric study of hybridization between Polystichum munitum and P.

imbricans was conducted by Mayer (1989) to test for hybridization, because

problematic individuals appeared to be intermediate between the two species. Plants

were scored for 12 macro- and microscopic characters. Specimens were then grouped

according to spore morphology as munitum, imbricans, or abortive. Plants with

abortive spores were morphologically intermediate in most respects, supporting the

hybridization hypothesis. Although the two parental species are morphologically

9
 distinct in the mixed populations, they are more similar when sympatric than

when allopatric. This convergence could be the result of genetic and/or phenotypic

responses to a common environment, or introgression.

Smith (1971) studied the systematics of the neotropical species of Thelypteris

section Cyclosorus, including morphology, chromosome numbers, hybridization,

ecology and distribution. He considered morphological characters of the rhizome,

stipe and rachis, blade, indumenta, sorus and indusium, spores and sporangia, and

gametophyte. Haufler et al. (1990) studied the Cystopteris tennesseensis complex,

combining morphometric analyses with surveys of chromosomal, isozymic, and

gametophytic features to differentiate the allotetraploid Cystopteris tennesseensis and

its putative diploid progenitors, C. bulbifera and C. protusa. They found that the

isozymic variation in the tetraploid parallels that of the diploids, implying that genetic

variability was introduced through recurrent hybridization.

Only in the past quarter of a century have the spores of ferns been used

extensively in elucidating systematic relationships. Fern spores display remarkable

variation and have been extremely helpful in systematic studies. In many cases

particular spore morphologies are distinctive for families or genera, or in some

instances individual species. Spore sizes and abortion have been useful in studies of

hybridization and polyploidy, and in some cases they have led to the discovery of

unknown reproductive variation in ferns. A broad detailed survey of spores is still

10
 needed to increase our understanding of variation patterns and to determine

which ones are meaningful taxonomically (Taylor and Mickel, 1974). Blasdell (1963)

and Moran (1982, cited in Haufler et al., 1990) noted the value of spore features for

identifying hybrids and for determining the ploidy level of specimens of Cystopteris.

Through analyses of meiotic chromosome behavior, Haufler et al. (1990) were able to

provide independent verification for Lovis’ (1977) caveat that it may not be

appropriate in Cystopteris to infer ploidy levels from spore measurements, while also

finding that spore size did correlate with several critical genetic features of the

species. They agreed with Lovis that spore size comparisons alone do not substitute

for direct chromosome analyses, but they found that spores can be extremely valuable

in surveying specimens rapidly once strong correlations between spore features and

genetic condition are established. In particular, they found that C. bulbifera spores

were larger than those of C. protusa, and spores of tetraploid C. tennesseensis showed

the further size increase commonly associated with higher ploidy levels.

Cytological studies

Although recent morphological data provide strong evidence for clarifying

relationships among members of the Polypodium virginianum complex, chromosomal

data continue to hold a central position in developing arguments for or against

11
 particular hypotheses about the evolution of Polypodium species (Haufler and

Wang, 1991).

The significance of chromosomes in the study of pteridophytes starts with

Manton’s book of 1950. There are still monographs and floras being published in

which chromosome numbers may not be listed. If they are listed, the information is

sometimes added much as one would characterize a species as either annual or

perennial. Stable chromosome numbers such as 29, 37 and 41 in ferns are not quite

susceptible to arithmetic manipulations. The most worthwhile attribute of these

numbers is to give unity to a genus. The first step in a study of the cytogenetics of a

species is the accurate determination of the chromosome number. Unfortunately, this

is as far as we have proceeded in many cases, and given the technical difficulties of

the material; even this has not been achieved in some cases (Britton, 1974). In

leptosporangiate ferns we have a valuable research tool in chromosome counts. Such

studies are of particular value in examining hybridization and in supporting genetic

and familial relationships (Taylor and Mickel, 1974).

In a series of reviews, Walker (1973, 1984) illustrated the value of

chromosomal data in understanding fern phylogeny. Because of the different base

numbers in ferns, chromosome counts can be used as evidence for generic and

familial circumscription. Within genera, analysis of polyploid series can help to

unravel the complexities resulting from reticulate evolutionary events. Finally, the

12
 formation of bivalents during meiosis in sterile hybrids can be used to test

hypotheses regarding the genetic similarity of the genomes contained in such hybrids.

Hybrid studies

Many examples of fertile hybrids are known in the ferns, but these usually

involve either apogamy or polyploidization (Conant and Cooper-Driver, 1980). Few

fertile diploid hybrids with regular reductional meiosis are known. A comprehensive

ecological study embracing all aspects of the life cycles of several related species is

needed, however, to increase our understanding of the ecological basis of local

endemism.

Conant (1983) did a revision of the genus Alsophila (Cyatheaceae) in the

Americas. His work included the Puerto Rican species, discussing their morphology,

cytology, speciation patterns, and hybrids. Conant concluded that it is difficult to

determine whether the hybrid plants encountered in the field are F1 or later generation

hybrids. Some tend to be rare and intermediate in morphology, suggesting they belong

to the first generation.

13
 Materials and Methods

Study area

Adiantum vivesii Proctor is only found on privately owned land located on the

north side of a ridge north of Puerto Rico Hwy. 119 at km.16.1 (18º24’13"N. Lat.,

66º54’17"W. Long.) in Barrio San Antonio of the Municipality of Quebradillas. The

elevation of the general area ranges from near sea level up to 198 m. Adiantum vivesii

is found in the limestone or karst region of northwestern Puerto Rico. This region is

underlain by limestone rocks of Oligocene or Miocene age. Topography varies

throughout the karst region, from extremely rugged to gentle rolling hills. Canyons,

sinkholes, and subterranean rivers, as well as these rolling hills, are the most common

features of the region. Soils in the limestone hills are generally shallow, well drained,

alkaline, and interspersed between limestone outcrops (Soil and Conservation Service,

1975 cited in U. S. Fish and Wildlife, 1994).

The population occurs within semi-evergreen seasonal forest in the subtropical

moist forest life zone (Ewel and Whitmore, 1973). This life zone, which covers 58%

of the total area in Puerto Rico and the U.S. Virgin Islands, is delineated by a mean

annual rainfall ranging from 1000 - 1100 mm to 2000 - 2200 mm and a mean

temperature between 18°C and 24°C (Ewel and Whitmore, 1973). Adiantum vivesii

14
occurs in a deeply shaded hollow at the base of a limestone hill in Quebradillas. The

ownership of the land is Mr. Ángel Rivera; he knows about the presence of Adiantum

vivesii in his property and he is willing to cooperate with Puerto Rican government

agencies and the U. S. Fish and Wildlife Service to protect this endemic fern.

Distribution and abundance

The population was located precisely on a topographic map (fig. 1). The extent

of the population was measured at the beginning of the study using a metric tape

measure. The measurement was repeated at the end of the study to identify any

changes. Careful excavations were performed around several apparent individuals of

Adiantum vivesii and A. tetraphyllum to determine the extent of connections among

them. Two small (ca. 10 cm) pieces of the rhizome were planted in soil from the area

to see whether they could form new plants.

Voucher specimens of the A. vivesii and A. tetraphyllum were deposited at the

MAPR herbarium of the University of Puerto Rico, Mayagüez Campus. Other

associated species were identified by Mr. Miguel A. Vives, but voucher specimens

were not collected. A comparison was made of the characteristics of sympatric

Adiantum species, based on Proctor (1989).

15
Morphometric analysis

Specimens were collected of haphazardly selected plants of Adiantum vivesii

and of A. tetraphyllum, which may represent its parent. Dried leaf material was used

to conduct a morphometric analysis. Twenty-one parameters were studied for each of

twenty blades of each species (table 1; fig.2). Spore measurements were also

obtained for A. vivesii and A. tetraphyllum, as described below.

The data obtained for the 22 characters for the two species studied were

analyzed with the MINITAB statistical software package (version 13.20 for Windows)

using one-way ANOVA.

Chromosome counts

Fertile material of Adiantum vivesii and A. tetraphyllum was field-fixed following the

standard protocols for chromosome preparations as modified by Haufler and Wang

(1991). Pinnae were removed from sporophytes and fixed in Farmer’s solution (a 3:1

mixture of 100% ethanol and glacial acetic acid) for at least 24 hours under

refrigeration. Groups of fixed sporangia were removed from the sori and placed on a

glass slide in a drop of saturated acetocarmine solution (carmine boiled in 45% acetic

acid and filtered to remove undissolved crystals); then they were teased apart using

steel dissecting needles. The slides were heated with an alcohol burner, then

16
 wrapped in a Kimwipe®, and thumb pressure was applied. The preparation

was scanned for cells at late diakinesis or early metaphase of meiosis. To improve the

preparation for the purposes of photographic documentation, the selected cells were

marked and the slide was supported by glass rods while inverted in a 45% acetic acid

solution. After the coverslip floated off, extraneous material was carefully removed,

an additional drop of stain was applied, the coverslip was replaced, and the

preparation was again squashed. The second squash usually puts all chromosomes in

a single plane and separates them maximally from each other (Haufler and Wang,

1991). Photographs were taken with color film (ASA 400) using an Olympus BHS

microscope and an Olympus PM-C35B photomicrography system. The photographs

were scanned in a Microtek E-3 Plus scanner, and processed using Adobe Photoshop

5.0 and Adobe Page Maker 6.5 programs and printed on high quality glossy paper on

an Epson Stylus Photo printer.

Light microscopy

Fresh or dried pinnules, sori, and sporangia of Adiantum vivesii and A. tetraphyllum

were observed under the the SZ-PT Olympus dissecting microscope at magnifications

of 100x to 630x. Photographs were taken, scanned, processed, and printed as

described in the previous section.

17
 Under the dissecting microscope, one frond of each species was examined for

areas where there were mature or well developed sporangia. A drop of PermountÒ

was placed in the center of a slide and the point of a dissecting needle was dipped into

the solution to wet the tip in order to provide a “glue” for picking up the spores. The

spores were spread on the slide; this procedure was repeated several times to

accumulate a hundred or more spores in the drop. The spores were covered by slowly

lowering a cover glass over them. Spore morphology was observed and spore size

was measured at 400x using a calibrated ocular micrometer and an Olympus BHS

microscope. The total length (including the perispore) of fifty spores of each species

was measured.

Scanning electron microscopy (SEM)

Rhizomes and fertile pinnules of A. vivesii were cleaned to remove surface

contaminants, cut into small pieces and fixed in FAA or Farmer’s solution for at least

4 hours. The fixative was discarded and two rinses with 70% ethanol were done. The

samples were dehydrated in an ascending ethanol series (75%, 85%, 95% for 10

minutes each, and 100% , 3 changes for 10 minutes each). The dehydrated specimens

were desiccated by transferring them to the prechilled critical point dryer chamber

(EMS 850 System) after quickly blotting off most of the alcohol. The specimens

were mounted onto aluminum stubs with double sticky tape. The samples were

18
 quickly transferred to a clean desiccator, which was tightly closed and stored at

least for two hours.

For spores of A. vivesii and A. tetraphyllum, a different procedure was required

in preparation for SEM studies. Spores were collected by allowing sporangia on fresh

leaves to dehisce in clean newspaper overnight. The spores were then submitted to an

acetolysis procedure, which were conducted under a fume hood. Mature spores of

Adiantum vivesii and A. tetraphyllum were placed in 1.5 ml microtest tubes, which

were placed inside a 100 ml beaker with ice water to reduce the rate of acetolysis.

(The acetolysis mixture is violently reactive to water, and the reaction is highly

exothermic.) Three drops of concentrated sulfuric acid and 27 drops of acetic

anhydride were added to the microtest tubes containing the spores. Only acetolysis-

resistant spore walls should persist, and it is inferred that these are chemically

composed of the substance called sporopollenin (Zetsche, 1932, cited in Pérez-Muñoz,

1991). Glacial acetic acid (0.75 ml) was added to the tube, which was centrifuged for

ten minutes, the time required for pellet formation. The pellet was washed twice with

glacial acetic acid and then with distilled water. Spores were transferred to an

aluminum stub with double sticky tape and air-dried.

All samples, including rhizomes, fertile pinnules, and spores were coated with

gold/palladium in a sputter-coater (Bio-Rad, model E-5000), and examined under the

JEOL 5410 LV Scanning Microscope to see whether all studied parts were well

19
formed. Photographs were taken with a Polaroid 4" x 5" camera with Polaroid 55

film. The photographs were scanned, processed and printed as described previously.

20
 Results

Distribution and abundance

Adiantum vivesii is restricted to a single location in Quebradillas. The extent

of the population at the beginning of the study ( March, 1999) was 17 m x 9 m; its

extent at the end of the study (March, 2000) was 21 m x 10 m. Seven or eight deep

excavations were made at different points throughout the entire population, and in

most of them rhizome connections between the apparent individuals were found (fig.

3A). One rhizome was photographed; it was about half a meter in length (fig. 3B).

The excavations made to the population of A. tetraphyllum showed that each apparent

plant was a single plant. Two 10 cm rhizome segments were collected, one in March

1999 and in March 2000. They were planted in soil from the area, and grew into

healthy plants within about three months that have survived to the present (fig. 3C-D).

Small numbers of fiddleheads and young blades were observed on each visit to the

study site. The production of sporangia was seen throughout the year. On all visits,

attempts were made to find fern gametophytes, but none were seen.

On one visit, in March of 1999, Miguel A. Vives prepared a list of

pteridophytes and flowering plants that were growing in the immediate area of A.

vivesii (table 2). In total, 25 species of pteridophytes and 74 species of

spermatophytes were found near the population. Eight species of the genus Adiantum

21
 were found in the vicinity. They were A. cristatum, A. fragile, A. latifolium, A.

melanoleucum, A. pulverulentum, A.tenerum, A. tetraphyllum and A. wilsonii. Of

these, only A. tetraphyllum was growing within the area occupied by A. vivesii. The

characteristics of A. vivesii and the eight sympatric Adiantum species were compared

(table 3). The most similar species to A. vivesii appear to be A. tetraphyllum and A.

latifolium. The wide-creeping rhizome and the 2-pinnate blade are features that A.

vivesii shared with both species. Adiantum latifolium have 6 to 16 pairs of pinnules

per pinnae, A. tetraphyllum have 18 to 27 and A. vivesii have 10 to 13 (table 3).

Morphometric analysis

The results of a morphometric analysis of Adiantum vivesii and A.

tetraphyllum based on 21 vegetative characters and one spore character (table 1) are

presented in figures 4 - 25. According to a series of one-way ANOVAs, significant

differences were observed between the two species for 16 of the vegetative characters

as well as spore size (table 4).

Chromosome counts

After two years of work and numerous attempts at acetocarmine squashes, no

preparations of A. vivesii sporogenous tissue were obtained from which its

22
chromosome number could be determined. Different stages of sporogenesis in the

spore mother cells were found and photographically documented for the species (figs.

26A-F). Although similar preparations of A. tetraphyllum were not photographed, it

appeared that the two species had similar chromosome numbers, and that in any case

A. vivesii does not have twice the chromosome number of A. tetraphyllum.

Light microscopy

The presence of sori on the margins of the pinnules with false indusia covering

the sporangia was observed throughout the year (fig. 27A). The sori of A. vivesii

contain both immature and mature sporangia at the same time (fig. 27B). A globose

appearance of the group of mature sporangia was observed in fresh pinnules of A.

vivesii (fig. 27C). Empty, open, mature sporangia were observed on many dried

fronds of A. tetraphyllum (fig. 27D).

Contraction of the annulus and rupture of lip cells occurred in mature

sporangia exposed to light under the dissecting microscope; variability in the size and

shape of the spores of A. vivesii was then evident (fig. 28A). The spores of A. vivesii

range from 10 to 19 mm in size and those of A tetraphyllum from 19 to 23 mm.

Under the microscope many of the spores of A. vivesii seemed blank, with a prune-

like surface due to the presence of depressions; many spores were black or with black

areas (figs. 28B-C). In Adiantum tetraphyllum, the spores were regularly shaped (figs.

23
 28D-E), and showed less variation in size than those of A. vivesii (Table 4;

figs. 25, 28D).

Scanning electron microscopy (SEM)

Rhizomes of A. vivesii appeared healthy and with many rhizoids important for

vegetative reproduction (fig. 29A). The sori with columns of immature sporangia on

the false indusium have a normal appearance (fig. 29B). The groups of unopened

mature sporangia had a grape-like appearance; also, the stalk and annulus seemed

normal (fig. 29C-D). The spores of A. vivesii have a spherical shape when observed

under the scanning electron microscope (fig. 30A). The spores of A. tetraphyllum

have a tetrahedral shape and are all about the same size (fig. 30B-C).

24
 Discussion

Adiantum vivesii may be a single plant. Hybrid plants, including ferns, may

have processes to reproduce and colonize new habitats; in this case A. vivesii used

rhizome proliferation to increase its area of occupation. Apparent new individuals

were seen, but they were all rhizome-connected (fig. 3A). Probably over time and due

to climatic factors such as rain, the decomposition of the rhizomes caused that the

connections were lost at a few points. The spreading of the species in the area is by

vegetative reproduction due to rhizome proliferation; long rhizomes were found in

many of the excavations (fig. 3B). It is concluded that the entire population is only

one individual that has proliferated by rhizomes. In contrast, the excavations made to

A. tetraphyllum demonstrated that each single plant do not have any rhizome

connection with another single plant of the same species in the area. The ability of the

rhizome to produce new plants was demonstrated (figs. 3C-D). The population is

healthy and, over one year of monitoring from March 1999 to March 2000, it

increased in size by about 37%. Throughout the year sporangia and spores were

produced, but signs of sexual reproduction as gametophytes or small plants were not

observed.

25
In the immediate area of the population of A. vivesii eight species of the genus

Adiantum were found, but only A. tetraphyllum grows within the A. vivesii population

(table 2). If A. vivesii is a hybrid, one of the seven species of Adiantum could be

another parental species.

Adiantum vivesii appears to be a distinct morphological taxon. The best

morphological features that can be used in the field to distinguish A. vivesii from A.

tetraphyllum are the number of lateral pinnae and the number of pinnules on each

lateral pinna, which are fewer in A. vivesii (figs. 8, 13). The leaf of A. vivesii is very

similar to that of A. tetraphyllum; for this reason Proctor thought that A. tetraphyllum

is the closest relative of this new species. The morphometric analysis showed great

variability in many features, but significant differences were not found at the level of

the entire frond. Petiole length, blade length and width, the blade length/width ratio

and the mean length of the lateral pinnae showed slight differences; many of the

values overlap between the two species (figs. 4-7, 9). Adiantum tetraphyllum showed

more within-species variability, apparently because the material used in the analysis

represented many individuals. It is believe that Adiantum vivesii showed less

variation because all the fronds belonged to the same individual (table 3, figs. 4-25).

Adiantum vivesii does not appear to be a good biological species, capable of sexual

reproduction. The presence of sori on the pinnules does not mean that the species

spores are capable of germinating and forming a new plant. The plant uses the

26
 rhizome for its vegetative reproduction and the entire population seems to be

connected by them (fig. 3A).

Examination of the hybrid and parental plants of Woodsia revealed

morphological intermediacy occuring in several characters on the hybrid plants

(Brooks, 1982). Palmer (1998) described morphological intermediacy in a hybrid of

the genus Cibotium, in comparison with its known parents.

The gross morphology helps in the identification of intermediacy between

parental species, but the spores must also be studied to know if the new plant is a

hybrid. Sterile intertaxa with defective spores are much more common than any other

types of hybrids (Wagner and Chen, 1965). Considering the high incidence of sterile

nothospecies among pteridophytes, the use of spore abortion as a means of

identification can be extremely important, especially where the parental species are

subtly differentiated on the basis of gross morphology.

Sterile hybrids tend to have a greater variation in spore size than normal

sexual species (Wagner and Chen, 1965). The greater variation in spore size in A.

vivesii is mainly produced by spore abortion (figs. 28A-C). Palmer (1998), in his

work with the Cibotium hybrid, found variability in spore morphology. Mayer (1989)

states that plants of the genus Polystichum that have abortive spores were

morphologically intermediate, supporting the hybridization hypothesis.

27
 The variability in spore morphology and spore size are supported by the studies

of Whittier and Wagner (1971) and Wagner et al. (1986). Normal species such as A.

tetraphyllum have at least some abortive spores (fig. 28E), but many fewer than in

sterile hybrids (Wagner and Chen, 1965). The forms of the spores of A. vivesii are

quite different because of the collapse of the exospore associated with the absence of

the protoplast. The empty spores were commonly filled with air producing optically

black areas within the exospore (fig. 28B-C).

The mature sori of A. vivesii (figs. 27C, 29B-D) seemed to be more compactly

constructed than those of A. tetraphyllum, with the sporangia appearing as more or

less globular objects tightly grouped together; this is consistent with the sorus of a

hybrid. Wagner and Chen (1965) found the abortion of sporangia and spores to be an

important tool in the detection of Dryopteris hybrids. Abortion can be characterized

as significant variation from the normal products of sporogenesis. Spore samples

showing exaggerated variability in spore size will often one or both show of the

following symptoms: Spore collapse may go hand in hand with loss of the protoplast.

Usually massive perisporial development occurs, together with a reduction in the size

of the protoplasts as compared to the parents (Wagner and Chen, 1965).

Wagner and Chen (1965) demonstrated that greater variation in spore size,

which is mainly produced by spore abortion, is characteristic of a sterile hybrid. The

phenomenon of spore abortion may be traced back to the process that produces the

28
 spores. The important point is that in hybrids the chromosomes tend to be

distributed irregularly to the young spores, and the spores differ genetically among

themselves.

Wagner and Chen (1965) proposed the following steps that are involved in the

detection of a hybrid found in nature: 1) Recognition that the majority or all the

characteristics of the plant in question are intermediate between those of two distinct

and well understood species. 2) Observation that the sori contain abortive sporangia

and spores. 3) Determination that the chromosomes, when studied in cytological

preparations under the compound microscope, do not behave normally in the process

of spore production. In this investigation only the last point was left unresolved. The

chromosome counts in both the immature sporangia and root tips do not present

clearly the chromosome number of the species.

In the case of Tectaria estremerana Proctor & Evans, which is believed to be

of autopolyploid origin, its chromosome number is twice that of its parent, Tectaria

cicutaria (L.) Copeland, Philipp. This is not the case for A. vivesii, where the

acetocarmine squashes did not differ substantially in appearance from those of A.

tetraphyllum and approximately the same number of chromosomes is suggested for

both species. It was demonstrated that A. tetraphyllum in Trinidad is diploid and

tetraploid, without morphological differences (Jermy and Walker, 1985, cited in

Moran et al., 1995).

29
 Adiantum vivesii is certainly a distinct morphological taxon that has

maintained itself and in fact is spreading vegetatively. Long-term monitoring of the

species is recommended, since not much difference in population size was observed

over a single year. Annual visits to the site would be reasonable to detect changes in

the size and status of the population. The habitat is safe for A. vivesii because there is

little or no human impact. Only a hurricane or major constructions (for example, a

road or housing development) could endanger the species’ stability.

As a species incapable of sexual reproduction, A. vivesii is not expected to

colonize other localities on its own. If it were deemed useful to pursue its

conservation ex situ, this could readily be done by rhizomes (fig. 3C-D). Other

suitable habitats should have the similar environmental conditions, and should

preferably share associated species (table 2) with the present site.

30
 Conclusions

1. Adiantum vivesii is restricted to its type locality in Quebradillas.

2. The population probably consists only of one individual with rhizome

proliferations.

3. Adiantum vivesii appears to be a distinct morphological taxon.

4. Adiantum vivesii does not appear to be a good biological species,

capable of sexual reproduction.

5. Adiantum vivesii does not appear to be a polyploid, at least of

Adiantum

tetraphyllum.

6. Adiantum vivesii appears to be sterile, and may be hybrid. One of its

parents may be A. tetraphyllum; the other is unknown.

31
 Recommendations

1. Further cytological studies of Adiantum vivesii.

2. Additional studies of spores obtained from various fronds.

3. Spore germination experiments.

4. Molecular systematics of Adiantum vivesii and all sympatric species of

Adiantum.

5. Vegetative propagation ex situ in similar habitats.

6. Long-term monitoring of the population.

32
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Wagner, W. H. and K.L. Chen. 1965. Abortion of spores and sporangia as a tool in
the detection of Dryopteris hybrids. Amer. Fern J. 55:9-29.

Wagner, W. H. Jr., F.S. Wagner and W.C. Taylor. 1986. Detecting abortive spores in
herbarium specimens of sterile hybrids. Amer. Fern J. 76: 129-140.

Walker, T. 1973. Evidence from cytology in the classification of ferns. In A. C.

Jermy, J. A. Crabbe, and B. A. Thomas ([eds.), The phylogeny and
classification of the fern. Academic Press, London.

Walker, T. 1984. Chromosomes and evolution in pteridophytes. In A. K. Sharma and

A. Sharma [eds.], Chromosomes in evolution of eukaryotic groups, vol. 2, 103-
141. CRC Press, Boca Raton, Florida.

White, R. A. 1974. Comparative anatomical studies of the ferns. Ann. Missouri Bot.
Gard. 61: 379-387.

Whittier, D.P. and W.H. Wagner Jr. 1971. The variation in spore size and
germination in Dryopteris taxa. Amer. Fern J. 61:123-127.

36
Woodland, D.W. 1997. Contemporary plant systematics. 2nd ed.. Andrews
University Press, Berrien Springs, Michigan. 581 pp.

Wylie, R. B. 1949. Variations in leaf structure among Adiantum pedatum plants

growing in a rock cavern. Amer. J. Bot. 36: 282-287.

37
Tables

38
Table 1. List of features included in morphometric analyses. Asterisks indicate those
illustrated in Figure 2.

1-9 FEATURES OF ENTIRE LEAF

* 1. Length of petiole (cm)
* 2. Length of blade (cm)
* 3. Width of blade (cm)
4. Length / width of blade
5. Number of lateral pinnae
* 6. Mean length of lateral pinnae (cm)
* 7. Mean width of lateral pinnae (cm)
8. Mean length / width of lateral pinnae
* 9. Distance between first and second lateral pinnae (cm)

10-13 FEATURES OF LATERAL PINNAE

10. Mean number of pinnules on lateral pinnae
* 11. Mean length of longest pinnule (cm)
* 12. Mean width of longest pinnule (cm)
13. Mean length / width of longest pinnule

14-21 FEATURES OF TERMINAL PINNA

* 14. Length of terminal pinna (cm)
* 15. Width of terminal pinna (cm)
16. Length / width of terminal pinna
* 17. Length of petiolule (cm)
* 18. Number of pinnules on terminal pinna
* 19. Length of largest pinnule on terminal pinna (cm)
* 20. Width of largest pinnule on terminal pinna (cm)
21. Length / width of largest pinnule on terminal pinna (cm)

22. SPORE FEATURE

22. Spore size (mm)

39
Table 2: Species associated with Adiantum vivesii, according to Miguel A. Vives.

SPECIES FAMILY SUBFAMILY

Pteridophyta

Cyathea arborea (L.) Sm. Cyatheaceae

Adiantum cristatum L. Polypodiaceae Adiantoideae

Adiantum fragile Sw. Polypodiaceae Adiantoideae

Adiantum latifolium Lam. Polypodiaceae Adiantoideae

Adiantum melanoleucum Willd. Polypodiaceae Adiantoideae

Adiantum pulverulentum L. Polypodiaceae Adiantoideae

Adiantum tenerum Sw. Polypodiaceae Adiantoideae

Adiantum tetraphyllum Humb. & Bonpl. ex Willd. Polypodiaceae Adiantoideae

Adiantum vivesii Proctor Polypodiaceae Adiantoideae

Adiantum wilsonii Hook. Polypodiaceae Adiantoideae

Arachniodes chaerophylloides (Poir.) Proctor Polypodiaceae Dryopteridoideae

Bolbitis pergamentaceae (Maxon) Ching Polypodiaceae Dryopteridoideae

Diplazium sp. Polypodiaceae Athyrioideae

Polypodium latifolium Vahl Polypodiaceae Polypodioideae

Polypodium phyllitidis L. Polypodiaceae Polypodioideae

Fadyenia hookeri (Sweet) Maxon Polypodiaceae Tectarioideae

Hypoderris brownii J. Smith Polypodiaceae Tectarioideae

Tectaria cicutaria (L.) Copel. Polypodiaceae Tectarioideae

40
Table 2 (cont.).

SPECIES FAMILY SUBFAMILY

Tectaria heracleifolia (Willd.) Underw. Polypodiaceae Tectarioideae

Tectaria trifoliata (L.) Cav. Polypodiaceae Tectarioideae

Thelypteris guadalupensis (Wikström) Proctor Polypodiaceae Thelypteridoideae

Thelypteris hildae Proctor Polypodiaceae Thelypteridoideae

Thelypteris pennata (Poir.) Morton Polypodiaceae Thelypteridoideae

Psilotum nudum (L.) P. Beauv. Psilotaceae

Anemia adiantifolia (L.) Sw. Schizaeaceae

Selaginella laxifolia Baker Selaginellaceae

Selaginella subcaulescens Baker Selaginellaceae

41
Table 2 (cont.).

SPECIES FAMILY

Spermatophyta

Justicia sp. Acanthaceae

Ruellia coccinea (L.) Vahl Acanthaceae

Comocladia glabra (Schultes) Spreng. Anacardiaceae

Anthurium crenatum (L.) Kunth Araceae

Didymopanax morototoni (Aubl.) Decne. & Planch Araliaceae

Prestoea borinquena (R. Grah.) Nichols. Asteraceae

Spathodea campanulata Beauv. Bignoniaceae

Tabebuia haemantha (Bert.) DC. Bignoniaceae

Tabebuia heterophylla (DC.) Britton Bignoniaceae

Ceiba pentandra (L.) Gaertn. Bombaceae

Bourreria domingensis (DC.) Griseb. Boraginaceae

Cordia laevigata (Lam.) Boraginaceae

Cordia sulcata DC. Boraginaceae

Guzmania sp. Bromeliaceae

Tetragastris balsamifera (Sw.) Oken Burseraceae

Cecropia schreberiana Mig. Cecropiaceae

Gyminda latifolia (Sw.) Urb. Celastraceae

42
Table 2 (cont.).

SPECIES FAMILY

Calophyllum calaba L. Clusiaceae

Clusia rosea Jacq. Clusiaceae

Diospyros sintenisii (Krug & Urb.) Standl. Ebenaceae

Sloanea amygdalina Griseb. Eleocarpaceae

Drypetes ilicifolia Krug & Urb. Euforbiaceae

Gymnanthes lucida Sw. Euphorbiaceae

Andira inermis (W.Wright) DC. Fabaceae

Casearia guianensis (Aubl.) Urb. Flacourtiaceae

Homalium racemosum Jacq. Flacourtiaceae

Gesneria pedunculosa (DC.) Fritsch Gesneriaceae

Ottoschulzia rhodoxylon (Urb.) Urb. Icacinaceae

Ocotea leucoxylon (Sw.) Mez Lauraceae

Mouriri helleri Britton Melastomataceae

Tetrazygia angustifolia (Sw.) DC. Melastomataceae

Guarea guidonea (L.) Sleumer Meliaceae

Inga vera Willd. Mimosaceae

Artocarpus altilis (Parkinson) Fosberg Moraceae

Pseudolmedia spuria (Sw.) Griseb. Moraceae

Trophis racemosa (L.) Urb. Moraceae

43
Table 2 (cont.).

SPECIES FAMILY

Ardisia obovata Desv. Myrsinaceae

Eugenia confusa DC. Myrtaceae

Eugenia ligustrina (Sw.) Willd. Myrtaceae

Myrcia paganii Krug & Urb. Myrtaceae

Pimenta racemosa (Mill.) J.W. Moore Myrtaceae

Psidium amplexicaule Pers. Myrtaceae

Syzygium jambos (L.) Alston Myrtaceae

Guapira fragans (Dum.-Cours.) Little Nyctaginaceae

Neea buxifolia (Hook. f.) Heimerl Nyctaginaceae

Oeceoclades maculata (Lindl.) Lindl. Orchidaceae

Roystonea regia (H.B.K.) O.F. Cook Palmae

Sabal causiarum (O.F. Cook) Becc. Palmae

Piper aduncum L. Piperaceae

Securidace virgata Sw. Polygalaceae

Coccoloba costata C. Wright Polygonaceae

Coccoloba diversifolia Jacq. Polygonaceae

Coccoloba pubescens L. Polygonaceae

Coccoloba pyrifolia Desf. Polygonaceae

Clematis dioica L. Ranunculaceae

44
Table 2 (cont.).

SPECIES FAMILY

Krugiodendron ferreum (Vahl) Urb. Rhamnaceae

Reynosia krugii Urb. Rhamnaceae

Cassipourea guianensis Aubl. Rhizophoraceae

Prunus myrtifolia (L.) Urb. Rosaceae

Coffea arabica L. Rubiaceae

Gonzalagunia sp. Rubiaceae

Neolaugeria resinosa (Vahl) Nicolson Rubiaceae

Randia aculeata L. Rubiaceae

Rondeletia inermis (Spreng.) Krug & Urb. Rubiaceae

Amyris elemifera L. Rutaceae

Pilocarpus racemosus L. Rutaceae

Zanthoxylum martinicense (Lam.) P. Wilson Rutaceae

Cupania americana L. Sapindaceae

Thouinia striata Radlk. Sapindaceae

Manilkara bidentata (A.DC.) Chev. Sapotaceae

Brunfelsia portoricensis Krug & Urb. Solanaceae

Symplocos martinicensis Jacq. Symplocaceae

45
 Table 3. Comparison of some characteristics of sympatric Adiantum species, based on Proctor (1989). “No
information” means that Proctor did not include it in his book.

Species Rhizome Blade Pinnae Pinnules Sori

Adiantum vivesii creeping 2-pinnate 2 or 3 pinnae 10-13 pairs up to 5 per pinnule
Adiantum fragile short 3-4 pinnate at base no information no information singly or in pairs
Adiantum latifolium wide-creeping 2-pinnate 1-4 pairs 6-16 pairs up to 12 per pinnule
Adiantum melanoleucum creeping 2-pinnate at base 2-8 pairs no information 2-6 per pinnule
Adiantum pulverulentum short-creeping 2-pinnate 3-9 pairs no information usually solitary

46
Adiantum pyramidale creeping 2-pinnate 2-11 pairs no information up to 9 per pinnule
Adiantum tenerum short-creeping 3-5-pinnate at base no information no information in pairs
Adiantum tetraphyllum creeping 2-pinnate 2-6 pairs 18-27 pairs no information
Adiantum wilsonii short- to long- 1-pinnate 1-3 pairs no information no information
creeping
 Table 4. Results of a morphometric analysis of Adiantum vivesii and A. tetraphyllum based on 21 vegetative characters and one
spore character. For each species, the mean and standard deviation (in parentheses) are given, together with the probability
value (P), obtained by a one-way ANOVA, and its level of significance: n.s. = not significant (P > 0.05),
∗ = significant (0.01< P ≤ 0.05), ∗∗ = highly significant (0.001< P ≤ 0.01), ∗∗∗ = very highly significant (P ≤ 0.001). For
the vegetative characters, n = 20 for each species; for the spore character, n = 50.

Character Adiantum vivesii Adiantum tetraphyllum Probability

Petiole length (cm) 31.8 (6.56) 30.5 (10.31) 0.637 n.s.
Blade length (cm) 23.8 (2.52) 24.5 (2.87) 0.374 n.s.

Blade width (cm) 28.3 (3.72) 28.3 (3.44) 0.997 n.s.

47
Blade length/width 0.86 (0.06) 0.87 (0.08) 0.588 n.s.

Number of lateral pinnae 2.60 (0.50) 5.10 (1.12) 0.000 ∗∗∗

Lateral pinnae mean length (cm) 14.4 (2.00) 14.9 (1.57) 0.408 n.s.
Lateral pinnae mean width (cm) 5.62 (0.79) 3.91 (0.67) 0.000 ∗∗∗
Lateral pinnae mean length/width 2.62 (0.47) 3.89 (0.65) 0.000 ∗∗∗
Distance between first and second lateral pinnae 2.63 (0.62) 2.05 (0.60) 0.004 ∗∗
Mean number of pinnules on lateral pinnae 9.94 (1.30) 20.0 (1.69) 0.000 ∗∗∗
 Table 4 (cont.).

Character Adiantum vivesii Adiantum tetraphyllum Probability

Mean length of longest pinnule on lateral pinna (cm) 3.26 (0.49) 2.20 (0.38) 0.000 ∗∗∗
Mean width of longest pinnule on lateral pinna (cm) 0.83 (0.13) 0.69 (0.10) 0.000 ∗∗∗
Mean length/width of longest pinnule on lateral pinna 3.98 (0.34) 3.20 (0.37) 0.000 ∗∗∗

Terminal pinna length (cm) 21.1 (2.28) 19.4 (1.98) 0.016 ∗

Terminal pinna width (cm) 7.38 (0.93) 4.96 (0.89) 0.000 ∗∗∗

48
Terminal pinna length/width 2.92 (0.45) 4.02 (0.67) 0.000 ∗∗∗
Petiolule length (cm) 1.24 (0.38) 0.76 (0.22) 0.000 ∗∗∗
Number of pinnules on terminal pinna 13.8 (1.20) 24.3 (1.81) 0.000 ∗∗∗
Largest pinnule length on terminal pinna (cm) 4.04 (0.47) 2.72 (0.45) 0.000 ∗∗∗
Largest pinnule width on terminal pinna (cm) 1.00 (0.13) 0.82 (0.12) 0.000 ∗∗∗

Largest pinnule length/width on terminal pinna 4.11 (0.39) 3.32 (0.37) 0.000 ∗∗∗
Spore size (µm) 13.7 (1.44) 20.8 (0.95) 0.000 ∗∗∗
Figures

49
Figure 1. Topographic map of barrio San Antonio, Quebradillas.
50
Figure 2. Leaf features included in morphometric analysis.
51
 A
A

C D

D
Figure 3A. Rhizome connections between the apparent individuals of Adiantum
vivesii.
3B. Rhizome of Adiantum vivesii about half a meter long.
3C. New plant of Adiantum vivesii, grown from rhizome collected in
March 1999.
3D. New plant of Adiantum. vivesii, grown from rhizome collected in
March 2000.
52
 60.0

50.0
petiole length (cm)

40.0

30.0

20.0

10.0

0.0
A. vivesii A. tetraphyllum
species

Figure 4. Dotplot of petiole length in Adiantum vivesii and Adiantum tetraphyllum.

Mean length of the petiole was 31.7 cm for A. vivesii and 30.4 cm for A. tetraphyllum.

53
 40.0

30.0
blade length (cm)

20.0

10.0

0.0
A. vivesii A. tetraphyllum
species

Figure 5. Dotplot of blade length in Adiantum vivesii and Adiantum tetraphyllum.

Mean blade length was 23.8 cm for A. vivesii and 24.5 cm for A. tetraphyllum.

54
 40.0

35.0

30.0
blade width (cm)

25.0

20.0

15.0

10.0

5.0

0.0
A. vivesii A. tetraphyllum
species

Figure 6. Dotplot of blade width in Adiantum vivesii and Adiantum tetraphyllum. Mean
blade width was 28.2 cm for A. vivesii and 28.2 cm for A. tetraphyllum.

55
 1.2

1.0
length/width ratio

0.8

0.6

0.4

0.2

0.0
A. vivesii A. tetraphyllum
species

Figure 7. Dotplot of blade length/width in Adiantum vivesii and Adiantum tetraphyllum.

The mean blade length/width was 0.86 for A. vivesii and 0.87 for A. tetraphyllum.

56
 9

8
number of lateral pinnae

0
A. vivesii A. tetraphyllum
species

Figure 8. Dotplot of number of lateral pinnae in Adiantum vivesii and Adiantum

tetraphyllum. The mean number was 2.6 for A. vivesii and was 5.1 for A.
tetraphyllum.

57
 20

18

16

14
length (cm)

12

10

0
A. vivesii A. tetraphyllum
species

Figure 9. Dotplot of mean length of lateral pinnae in Adiantum vivesii and Adiantum
tetraphyllum. The mean length was 14.4 cm for A. vivesii and 14.9 cm for A.
tetraphyllum.

58
 9.0

8.0

7.0
width (cm)

6.0

5.0

4.0

3.0

2.0

1.0

0.0
A. vivesii A. tetraphyllum
species

Figure 10. Dotplot of mean width of lateral pinnae in Adiantum vivesii and Adiantum
tetraphyllum. The mean width was 5.6 cm for A. vivesii and 5.7 cm for A.
tetraphyllum.

59
 7

6
length/width ratio

0
A. vivesii A. tetraphyllum
species

Figure 11. Dotplot of mean length/width of lateral pinnae in Adiantum vivesii and
Adiantum tetraphyllum. The mean length/width was 2.6 for A. vivesii and 3.9 for A.
tetraphyllum.

60
 4.0

3.0
distance (cm)

2.0

1.0

0.0
A. vivesii A. tetraphyllum
species

Figure 12. Dotplot of distance between first and second lateral pinnae in Adiantum
vivesii and Adiantum tetraphyllum. The mean distance was 2.6 for A. vivesii cm and
2.0 cm for A. tetraphyllum.

61
 24.0
22.0
20.0
number of pinnules

18.0
16.0
14.0
12.0
10.0
8.0
6.0
4.0
2.0
0.0
A. vivesii A. tetraphyllum
species

Figure 13. Dotplot of mean number of pinnules on lateral pinnae in Adiantum vivesii
and Adiantum tetraphyllum. The mean number was 9.9 for A. vivesii and 19.9 for A.
tetraphyllum.

62
 5.0

4.5

4.0

3.5
length (cm)

3.0

2.5

2.0

1.5

1.0

0.5

0.0
A. vivesii A. tetraphyllum
species

Figure 14. Dotplot of mean length of longest pinnule on lateral pinnae in Adiantum
vivesii and Adiantum tetraphyllum. The mean length was 3.2 cm for A. vivesii and 2.2
cm for A. tetraphyllum.

63
 1.5
1.4
1.3
1.2
1.1
1.0
width (cm)

0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
A. vivesii A. tetraphyllum
species

Figure 15. Dotplot of mean width of longest pinnule on lateral pinnae in Adiantum
vivesii and Adiantum tetraphyllum. The mean width was 0.83 cm for A. vivesii and
0.68 cm for A. tetraphyllum.

64
 5.0

4.0
length/width ratio

3.0

2.0

1.0

0.0
A. vivesii A. tetraphyllum
species

Figure 16. Dotplot of mean length/width of longest pinnule on lateral pinnae in Adian-
tum vivesii and Adiantum tetraphyllum. The mean ratio was 3.9 for A. vivesii and 3.2
for A. tetraphyllum.

65
 28.0

24.0

20.0
length (cm)

16.0

12.0

8.0

4.0

0.0
A. vivesii A. tetraphyllum
species

Figure 17. Dotplot of length of terminal pinnae in Adiantum vivesii and Adiantum
tetraphyllum. The mean length was 21.1 cm for A. vivesii and 19.4 cm for A.
tetraphyllum.

66
 10.0

8.0
width (cm)

6.0

4.0

2.0

0.0
A. vivesii A. tetraphyllum
species

Figure 18. Dotplot of width of terminal pinnae in Adiantum vivesii and Adiantum
tetraphyllum. The mean width was 7.4 cm for A. vivesii and 4.9 cm for A.
tetraphyllum.

67
 6
length/width ratio

0
A. vivesii A. tetraphyllum
species

Figure 19. Dotplot of length/width of terminal pinnae in Adiantum vivesii and Adiantum
tetraphyllum. The mean length/width was 2.9 for A. vivesii and 4.0 for A.
tetraphyllum.

68
 2.8

2.4

2.0
length (cm)

1.6

1.2

0.8

0.4

0.0
A. vivesii A. tetraphyllum
species

Figure 20. Dotplot of petiolule length in Adiantum vivesii and Adiantum tetraphyllum.
The mean length was 1.23 cm for A. vivesii and 0.75 cm for A. tetraphyllum

69
 28

24
number of pinnules

20

16

12

0
A. vivesii A. tetraphyllum
species

Figure 21. Dotplot of number of pinnules on terminal pinnae in Adiantum vivesii and
Adiantum tetraphyllum. The mean number was 13.8 for A. vivesii and 24.3 cm for A.
tetraphyllum

70
 6.0

4.0
length (cm)

2.0

0.0
A. vivesii A. tetraphyllum
species

Figure 22. Dotplot of length of largest pinnule on terminal pinnae in Adiantum vivesii
and Adiantum tetraphyllum. The mean length was 4.0 cm for A. vivesii and 2.7 cm in
A. tetraphyllum.

71
 2.0
width (cm)

1.0

0.0
A. vivesii A. tetraphyllum
species

Figure 23. Dotplot of width of largest pinnule on terminal pinnae in Adiantum vivesii
and Adiantum tetraphyllum. The mean width was 0.99 cm for A. vivesii and 0.82 cm
for A. tetraphyllum

72
 6.0
length/width ratio

4.0

2.0

0.0
A. vivesii A. tetraphyllum
species

Figure 24. Dotplot of length/width of largest pinnule on terminal pinnae in Adiantum

vivesii and Adiantum tetraphyllum. The mean length/width was 4.1 for A. vivesii and
3.3 for A. tetraphyllum

73
 24

20

16
spore size

12

0
A. vivesii A. tetraphyllum
species

Figure 25. Dotplot of spore size (mm) in Adiantum vivesii and Adiantum tetraphyllum.
The mean spore size was 13.6 mm for A. vivesii and 20.8 mm for A. tetraphyllum.

74
A B 0.01 mm
0.05 mm

0.02 mm

C 0.03 mm
D

0.01 mm
E F
0.02 mm
Figure 26. Stages of meiosis in sporogenous tissue in Adiantum vivesii.

75
A B

C D

Figure 27A. Adiantum vivesii pinnule with sori.

27B. Adiantum vivesii mature sporangia.

27C. Mature sporangia of Adiantum vivesii.

27D. Mature opened sporangia of Adiantum tetraphyllum

76
 A 0.01mm

B 0.15 mm C 0.02 mm

D 0.02 mm
E 0.03 mm

Figure 28A. Contraction of annulus and lip cells of Adiantum vivesii.

28B. Abortive spores of Adiantum vivesii (20x).

28C. Close-up of abortive spores of Adiantum vivesii (40x).

28D. Adiantum tetraphyllum normal spores (20x)

28E. One abortive spore in Adiantum tetraphyllum.

77
A B

C D

Figure 29A. Scanning photograph of rhizome in Adiantum vivesii.

29B. Scanning photograph of immature sporangia in Adiantum. vivesii.

29C. Scanning photograph of mature sporangia in Adiantum vivesii

29D. Close-up of a scanning photograph of mature sporangia in Adiantum

vivesii.

78
 A

B C

Figure 30A. Scanning photograph of the spores of Adiantum vivesii.

30B. Scanning photograph of Adiantum tetraphyllum spores.

30C. Close-up of a scanning photograph of Adiantum tetraphyllum spores.

79