[page 13↓]

2  Materials and Methods

2.1 Materials

This study is primarily based on the Cuban and Antillean species of the genus Pleurothallis (sensu Luer 1986b). For palynological and phytogeographical studies, however, taxa from other pleurothallid genera were added to provide a broader view on these orchids. Pleurothallis ekmanii Schltr., Pleurothallis excentrica (Luer) Luer (bas. Octomeria excentrica Luer) and Pleurothallisflabelliformis’ nom. prov. (bas. Octomeria prostrata H. Stenzel), three species of uncertain phylogenetic position, were treated under the generic name Pleurothallis, which, unfortunately, made the employment of an unpublished name unavoidable.

Material was collected in Cuba, Jamaica, the Dominican Republic, and Puerto Rico during the years 1997 - 2001. The vast majority of the samples was gathered during a 6-month stay in Cuba in 1998 (Tab. 1), another in 1999, and a third one of 3 months in 2000 in Jamaica and Puerto Rico. Initially, localities which should be visited were chosen from herbarium data and from information found in literature. Additional suggestions came from Dr. M. A. Díaz (HAJB), Dr. H. Dietrich (JE) and Dr. A. Urquiola Cruz (HPPR) resulting from former field trips within the Flora de Cuba Project (hereafter FCP).

Fig. 2: Collecting areas in Cuba.

1 – Guanahacabibes; 2 –Sierra de los Órganos; 3 – Cajálbana; 4 – Pan de Guajaibón; 5 – Taco Taco, Rangel; 6 – Montañas de Trinidad; 7 – Sierra de Banao; 8 – Turquino Massif; 9 – Guisa, Victorino; 10 – Gran Piedra; 11 – Palenque, Bernardo, Pico Galán; 12 – Sierra de Imías; 13 – Meseta del Toldo; 14 – Sierra del Cristal; 15 – Sierra de Nipe.

The majority of the more than 1300 vouchers collected belongs to the family Orchidaceae, with almost 500 representing taxa of the subtribe Pleurothallidinae. Collecting and transfer of the material took place in accordance with Cuban law and CITES criteria. In general, collecting of entire populations was avoided. In concordance with the rules of the FCP, herbarium material belonging to Pleurothallis will be split between HAJB and JE. [page 14↓]Holotypes and vouchers consisting of single specimens will be deposited in HAJB in any case. Material in spirit, CARNOY as well as silica dried samples belong to the personal collections of the author. All material that does not belong to Pleurothallis will be housed in HAJB.

Tab. 1. Survey of the field work in Cuba.

Province

Area

Date

collections
H. Stenzel

PR

Sierra del Rosario

III-1998

400-414

PR

El Moncada; Pan de Guajaibón; Cajálbana

IV-1998

415-429

Ho

Sierra de Nipe

IV-1998

430-493

Ho

Sierra de Moa: Reserva de Jaguaní

V-1998

494-503

Ho

Sierra del Cristal: Río Levisa

V-1998

504-547

Ho

Sierra de Moa,:between El Piloto and El Toldo

VI-1998

558-601

Ci, SS

Sierra de Escambray

VII-1998

602-618

Gr

Sierra Maestra: Victorino

VIII-1998

619-640

Gr, SC

Sierra Maestra: Turquino Massif

VIII-1998

641-663

SC

Santiago de Cuba: Gran Piedra

VIII-1998

664-680

PR

Sierra del Infierno

III-1999

710-716

PR

Mantua: La Cana

III-1999

716-721

Ho

Sierra de Nipe

IV-1999

722-743

Ho

Sierra del Cristal

IV-1999

744-778

Gr, SC

Sierra Maestra: Turquino Massif

IV-1999

780-839

Gu

Palenque; Piedra La Vela

IV,V1999

839-849

Gu

Yateras: Pico Galán

V-1999

850-874

Gu

Yateras: Bernardo

V-1999

875-876

Gu

Sierra de Imías: Los Calderos

V-1999

877-880

Gu

Sierra de Imías: La Yamagua

V-1999

881-916

PR

Sierra de Quemado: El Moncada

VI-2001

1100-1112, 1139-1166, 1211-1249

PR

Sierra de Cajálbana, S slope

VI-2001

1113-1138

 

Guanahacabibes: Cabo Corrientes

VI-2001

1167-1174, 1210

PR

Guanahacabibes: road to Cabo San Antonio

VI-2001

1175-1209, 1323

PR

Pan de Guajaibón

VI-2001

1250-1299

PR

Sierra de Cajálbana, N slope

VI-2001

1300-1322

Besides these personal collections, material of the following herbaria was examined: AJBC, BSC, HAC, HAJB, HPPR, JE, B, BM, BR, DUKE, G, GH (AMES), GOET, IJ, K (including K-L), NY, S, UPRRP, and W. Xerocopies or scanned images of material of the following herbaria was included, too: B-W (IDC-copies), GH (AMES), M, S.

Collection of plant material

Plants were dried in a portable press in the field. To preserve the details of the delicate and fragile generative organs, part of the material was conserved in spirit.

As far as possible, each voucher was completed by information on

  1. plant characteristics in vivo (colours, scents, growing habit, inflorescence and flower posture, flower characteristics);
  2. population characteristics (number of individuals, ~ flowering individuals);
  3. the locality (geographic position with GPS data), altitude, elevation above ground in the case of epiphytic populations (higher levels of trees etc. were examined by [page 15↓]eye and binoculars), cardinal point of the growing site on bark or rock, geological and edaphic characteristics;
  4. bio-ecological data (phorophytes, accompanying flora, vegetation type, observations of potential pollinators).

Mature flowers for palynological studies were separately dried in paper envelopes.

Slide images were taken of the natural environment and the collected plants.

Samples for molecular analyses were taken from fruits as well as from young and mature leaves. They were externally cleaned with ethanol (70%), cut into pieces to facilitate dehydration and stored in perforated paper envelopes in silica gel. In the first run (1998) desiccated material was stored only at (subtropical) room temperature. Later, material was maintained under cool conditions as soon as the pieces were dry. Even small intervals of time (from one month on!) under subtropical conditions led to severe deterioration concerning quality and amount of extractable DNA.

2.2  Methods

Descriptions and drawings

Descriptions were made including all material examined. Since the majority of the species could be collected in flowering condition, flower sections were carried out based mostly on personal spirit collections of the author. In most cases type material was not dissected, for many of the unique historical collections by Jacquin, Wright or Schlechter, are poor in generative material. Dry herbarium material was rehydrated in boiling water. Spirit material was dissected without further preparation. Pencil drawings were later copied with ink and scanned at high resolution. Scanned images were then arranged in position and size and provided with scales. All drawings are done by the author.

Reproductive biology

Phenological information was gathered from observations in the field and from herbarium material.

Ecological and phytogeographical patterns

General phytogeography – Data of distribution was collected from herbarium sheets in the herbaria mentioned above and was completed by information from the following sources: Ackerman 1995, 1997; Adams 1971, 1972, & pers. comm.; Ames & Correll 1952; Carnevali & al. 2001; Correll 1965; Dix & Dix 2000; Dietrich s. p.8; Dod 1974, 1984b, 1986b, 1989b; Foldats 1970; Dressler 1993b; Garay & Sweet 1974; Gloudon & Tobisch [page 16↓]1995; Hamer 2001; Luer 1975a, 1975b, 1990, 1998b, 1998c, 1999a, 1999b, 2000; MacLeish & al. 1995; Nir 2000; Stehlé 1939; Williams 1951; Williams & Allen 1946; Williams & al. 1980. Additional information from the NYBG West Indian Orchidaceae Specimens database (http://www.nybg.org/bsci/hcol/wior/orchidchecklist.html) and the MBG W3TROPICOS database (http://mobot.mobot.org/W3T/Search/vast.html) was taken into consideration.
This analysis is aimed at the comparison of the Greater Antilles islands with adjacent Lesser Antilles and continental areas. It includes all species of Pleurothallis found in the Greater Antilles. Variables are: West Cuba, Central Cuba, East Cuba, Jamaica, Hispaniola, Puerto Rico, Lesser Antilles, Central America, and South America.

Vertical distribution – Data of the vertical distribution of Cuban Pleurothallis species was gathered from personal observations and herbarium material. Occurrence of taxa was recorded in altitudinal belts of 100 m. Gaps within the vertical distribution were interpolated, which did not alter the graph qualitatively.

Geology – The distribution of the Cuban species of Pleurothallis in vegetation on three distinct types of rock (limestone, magmatic or volcanic rock, serpentine) was analysed. Petrologic (type of rock) data was obtained from personal observations and information on herbarium sheets. These facts were compared with Borhidi (1996), Samek (1973). Serpentine, though representing only one of the ultrabasic rock types is used in the broader sense of the latter hereafter. In the case of volcanic rock, the data may comprise actually a variety of types, which could not be differentiated. In fact, this category is more a negative circumscription: in no case it does contain stands, neither on ultrabasic rock nor on limestone. These stands are limited to the Sierra Maestra chain.

Veget ation types – Data concerning vegetation types are based on personal observations and information on herbarium sheets. These were compared with Samek (1973), Borhidi (1996), and Capote & Berazaín (1984). Assigning epiphytes to the vegetation types suggested by these authors turned out to be problematic occasionally. First, herbarium data is very scarce. Pleurothallis is distributed almost exclusively in colline to montane areas, where vertically and horizontally interlocked vegetation types can change within a few meters. Information from herbarium sheets, apart from their second hand character, are often too general. Second, classification of vegetation types may differ substantially between authors. Three problems occurred:1) types were partially incomparable because of inconsistent methods used by the authors and due to inaccurate descriptions, 2) there were cases of general misapplication of terms, and 3) some types turned out to be useless in classifying epiphytes (Pleurothallidinae). The latter was detected only in the course of the present study and will be discussed in detail later.
In the present study vegetation types sensu (Borhidi 1996) were used, however, with [page 17↓]some changes. Considering the differences that exist in the usage of even such common terms as “elfin” and “montane” (Capote & Berazaín 1984; Borhidi 1996) it seems reasonable to define the terms used in this study.

Computing and representation of the data in diagrams

Dendrograms – Data was set up in matrices in which species were coded as present (1) or absent (0). Species represented by single collections (Pleurothallis appendiculata, P. ghiesbreghtiana, P. aristata) or questionable classification (P. murex was described from sterile material) are not included in the matrices that were used to show correlations. To assess and show groupings of variables, Maximum Parsimony (MP) analyses were conducted. Dendrograms were initially rooted with a hypothetical outgroup with all species coded absent (Lundberg rooting), which was abandoned in a second run (see p. 78). Computation was carried out with the PHYLIP package (Felsenstein, J. 1989: PHYLIP (Phylogeny Inference Package) version 3.5c). This algorithm follows largely PAE (parsimony analysis of endemicity) introduced by Rosen & Smith (1988). See Trejo-Torres & Ackerman (2001) for a detailed discussion of this method.

3-dimen sional diagrams – Most ecological variables, e.g. types of rock, vegetation, and vertical amplitude, were examined as to their correlation with overall horizontal distribution of the taxa. Although the same data was statistically evaluated, too, 3-dimensional diagrams were chosen for presentation, because of the intriguing and lucid way they convey information. In order to present the diagram in a clearly arranged manner, species [page 19↓]were classified according to their occurrence in n locations (i.e. frequency) in the Caribbean (6 locations: West Cuba, Central Cuba, East Cuba, Jamaica, Hispaniola, Puerto Rico) and adjacent areas (GA-SA: Lesser Antilles, Central America, South America). The latter, because of its vast area, does not represent a 7th location. Thus, species of distribution outside the Greater Antilles, may or may not be present in all 6 inner Antillean localities. Again, in order to avoid overloading the diagrams, ecological amplitudes were classified, too, usually in three units representing the growing amplitude. Hence, horizontal distribution is growing on the x-axis from left to right and the ecological amplitude from the foreground towards the background (see Fig. 56 e.g.).

Statistics (correlation) – As just mentioned, an association between horizontal distribution and the ecological amplitude was examined. Species were grouped according to the number of areas they occur in (see previous paragraph for a definition). All correlations were performed with the SPSS 10.0 package, rs represents the Spearman’s rank correlation coefficient (Sokahl & Rohlf 1995), N the number of species, P is the significance value. The significance criterion for all tests was set at alpha=0.05, therefore P-values smaller than P=0.05 are considered as significant.

Palynology

The material included in the study of pollen morphology comprises ~100 samples of 72 pleurothallid and 2 other orchidaceous species (Tab. 2). The latter were used in the molecular study by (Pridgeon & al. 2001) as outgroup taxa. All but three Cuban species (Pleurothallis caymanensis, P. oricola, P. nummularia) currently treated as Pleurothallis could be included. With the exception of Brachionidium parvum and Pleurothallis aristata, from Puerto Rico, all Cuban taxa were represented by one local voucher at least. The Cuban endemic Pleurothallis grisebachiana, a widespread species variable in size and flower coloration, is represented by 10 samples from 9 different localities (Fig. 45), to test the degree of variation in pollen morphology. The same applies to Pleurothallis mucronata (Fig. 46) and P. ekmanii (Fig. 47) three Cuban localities each), P. ghiesbreghtiana (three samples from Central America, and one each from Cuba, Jamaica and Puerto Rico) and Pleurothallis ruscifolia (Jacq.) R. Br. (5 samples). The only subgenus of Pleurothallis that is endemic in the Antilles (~10 species, Luer 2000), subg. Antilla Luer, is represented by 7 species with 3 being restricted to Cuba.
Antillean material from closely related genera (Brachionidium, Lepanthes, Lepanthopsis, Platystele, Stelis, Trichosalpinx, Zootrophion) as well as from presumed sister or outgroup taxa in previous cladistic studies of Pleurothallidinae, Arpophyllum (Neyland & al. 1995, Pridgeon & al. 2001) and Dilomilis (Pridgeon & al. 2001) was included for comparative purposes.


[page 20↓]

All dry specimens were rehydrated for 1-5 min in hot water. All spirit material (Stenzel 482, Stenzel 765, Stenzel 1298) was dissected directly. Pollinia were removed, mounted on double tape and dried at room temperature. Pollinia were finally gold sputtered 100 s at 30 mA and a distance of 6 cm from the electrode.

Pollinia were examined with a JEOL SM 6300 SEM at the Palynological Laboratory of the Natural History Museum Stockholm and at the Museum of Natural History Berlin. Working data is as follows: Acceleration voltage 15 kV, probe current 3x10-11 A, distance 8-12 mm. Since the upper half of the pollinium tends to be more stable (Schill & Pfeiffer 1977) and generally free of elastoviscin (Stenzel 2000), data of this area was used for comparison. Caudicular regions were documented as well. If not indicated otherwise, photos show the entire pollinarium and details on the side of the upper pollinium which faces the tapetum. Pollen terminology, if adequate, was applied according to Punt & al. (1994). For description of additional terms see glossary (p. 170).


[page 21 - 22↓]

Tab. 2: Plant material used for palynological studies (species present in Cuba in bold type).

Species

Voucher

Country* & Locality**

Arpophyllum giganteum Hartw. ex Lindl.

Hort. Kew. (1932-18801)

?

?

Brachionidium parvum Cogn.

Stenzel 1005

PR

Carribean Nat. Forest, Pico de Este, W slope

Dilomilis elata (Benth. & Hook.) Summerhayes

Stenzel 765

CU

Ho: Sierra del Cristal, S slope, near summit

Lepanthes diaziae Luer

Stenzel 480

CU

Ho: Sierra de Nipe, Cayo Las Mujeres, W slope

Lepanthes dorsalis Schltr.

Stenzel 481

CU

Ho: Sierra de Nipe, Arroyo Woodfred, S bank

Lepanthes dressleri Hesp.

Stenzel 410

CU

PdR: mogotes E of Río Taco Taco, S of El Rangel

Lepanthes fulva Lindl.

Stenzel 624

CU

Gr: Sierra Maestra, Bayamo, Victorino, El Gigante, summit

Lepanthes melanocaulon Schltr.

Stenzel 457

CU

Ho: Sierra de Nipe, Cayo Las Mujeres (Alto de la Torre)

Lepanthes obliquilobia Hesp.

Stenzel 401

CU

PdR: Sierra del Rosario, Mogote Peña Blanca

Lepanthes silvae Dietrich

Stenzel 561

CU

Ho: Sierra de Moa, Meseta del Toldo, along headwaters of Río Piloto

Lepanthes trichodactyla Lindl.

Stenzel 72

CU

Gr: Sierra Maestra, Turquino Massif, below La Aguada de Joaquín

Lepanthes trichodactyla Lindl.

Stenzel 447

CU

Ho: Sierra de Nipe, La Mensura, north slope

Lepanthes turquinoensis Schltr.

Stenzel 76

CU

Gr: Sierra Maestra, Turquino Massif, below La Aguada de Joaquín

Lepanthopsis microlepanthes (Griseb.) Ames

Stenzel 664

CU

SC: Sierra Maestra, Santiago de Cuba, La Gran Piedra

Lepanthopsis pygmaea C. Schweinf.

Stenzel 632

CU

SC: Sierra Maestra, Bayamo, Victorino, El Gigante, summit

Platystele hyalina H. Stenzel

Stenzel 569

CU

Ho: Sierra de Moa, Meseta del Toldo, NE of abandoned miner’s camp El Piloto, along creek

Platystele ovalifolia (Focke) Garay & Dunsterv.

Stenzel 860

CU

Gu: Yateras, N ridge of Pico Galán

Pleurothallis alpestris (Sw.) Lindl.

Stenzel 959

JA

John Crow Mountains, Cuna Cuna Pass

Pleurothallis appendiculata Cogn.

PFC 63808 ex HAJB

CU

Gr: Sierra Maestra, Buey Arriba, near Barrio Nuevo

Pleurothallis aristata Hooker

Stenzel 1000

PR

Río Grande, Río Sonadora, 1,5 km upstream from Rd. 186

Pleurothallis aristata Hooker

Luer & Luer 12227

SU

Montagne de Kaw

Pleurothallis bissei Luer

Stenzel 482

CU

Ho: Sierra de Nipe, Woodfred, along N bank of creek

Pleurothallis brighamii S. Wats.

Stenzel 473

CU

Ho: Sierra de Nipe, behind EIIM station and Sabina trail

Pleurothallis brighamii S. Wats.

Stenzel 505

CU

Ho: Sierra del Cristal, lower Río Levisa, gallery forest

Pleurothallis caymanensis C.D.Adams

Stenzel 1209

CU

PR: Guanahacabibes, Barra de la Sorda, E from lighthouse Roncali

Pleurothallis claudii D.D.Dod

Dod 527 ex NY09199

DR

S side of the road Hondo Valle, towards Sierra de Neiba, km 204, km 190, 1300m

Pleurothallis cordatifolia Dod

Stenzel 192

DR

Sierra de Bahoruco, near border with Haiti, above military base El Aguacate

Pleurothallis corniculata (Sw.) Lindl.

Stenzel 472

CU

Ho: Sierra de Nipe, behind EIIM station and Sabina trail

Pleurothallis corniculata (Sw.) Lindl.

Stenzel 477

CU

Ho: Sierra de Nipe, base of Salto de Guayabo, N bank

Pleurothallis curtisii D.D.Dod

ex NY 60792

  

Pleurothallis delicatula Lindl.

Stenzel 958

JA

John Crow Mountains, Cuna Cuna Pass

Pleurothallis denticulata Cogn.

Stenzel 644

CU

SC: Sierra Maestra, Turquino massif, ascent to La Aguada de Joaquín, above Loma del León

Pleurothallis denticulata Cogn.

Wright "bbbb" ex W 42704

CU

“Oriente, prope Monte Verde”

Pleurothallis domingensis Cogn.

Stenzel 662

CU

SC: Sierra Maestra, Macizo del Turquino, Pico Real

Pleurothallis domingensis Cogn.

Lippold s.n. ex JE 16133

CU

Gr: S. Maestra, Pico Bayamesa, N slope

Pleurothallis domingensis Cogn.

Stenzel 197

DR

Sierra de Neiba, summit of ridge

Pleurothallis domingensis Cogn.

Tuerckheim 3331 ex NY 60844

DR

Constanza

Pleurothallis domingensis Cogn.

Alain 18269 ex NY 60836

DR

Constanza, Alto Casabito

Pleurothallis ekmanii Schltr.

Stenzel 517

CU

Ho: Sierra del Cristal, south side of Pico Cristal, upper Río Levisa, epiphytic in dwarf gallery forest

Pleurothallis ekmanii Schltr.

Stenzel 762

CU

Ho: Sierra del Cristal, south side of Pico Cristal, trail to the peak, elfin thicket, on rocks

Pleurothallis ekmanii Schltr.

Stenzel 573

CU

Ho: Sierra de Moa, Meseta del Toldo, epiphytic dwarf gallery forest along headwaters of Río Piloto

Pleurothallis excentrica (Luer) Luer

Stenzel 466

CU

Ho: Sierra de Nipe, La Mensura, north slope, broadleaf forest

Pleurothallis flabelliformis ’ H. Stenzel

Stenzel 507

CU

Ho: Sierra del Cristal, lower Río Levisa, gallery forest

Pleurothallis formondii D.D.Dod

ex NY 9205

  

Pleurothallis gelida Lindl.

P. Herrera ex HAC 3356

CU

SC: La Gran Piedra

Pleurothallis gelida Lindl.

Endres ex K

CR

?

Pleurothallis gemina H. Stenzel

Stenzel 464

CU

Ho: Sierra de Nipe, La Mensura, north slope

Pleurothallis ghiesbreghtiana A. Rich. & Galeotti

Hort. Kew. (1999-2869)

EC

?

Pleurothallis ghiesbreghtiana A. Rich. & Galeotti

Hort. Kew. (1968-22804)

NI

?

Pleurothallis ghiesbreghtiana A. Rich. & Galeotti

S.A. Marshall & D. A. Neill 6685 ex B

NI

Chontales

Pleurothallis ghiesbreghtiana A. Rich. & Galeotti

Stenzel 1298

CU

PR: Pan de Guajaibón, summit area

Pleurothallis ghiesbreghtiana A. Rich. & Galeotti

Stenzel 967

PR

Maricao, Maricao Forest Reserve, Monte de Estado, Rt. 120, km 16.85

Pleurothallis ghiesbreghtiana A. Rich. & Galeotti

“Hort. Lodd. s.n., 1824” ex K

JA

?

Pleurothallis grisebachiana Cogn.

Stenzel 489

CU

Ho: Sierra de Nipe, headwaters of Río Piloto, gallery forest, serpentine

Pleurothallis grisebachiana Cogn.

Stenzel 496

CU

Ho: Sierra de Moa, Jaguaní Reserve, Arroyo Prieto, serpentine?

Pleurothallis grisebachiana Cogn.

Stenzel 504, 538

CU

Ho: Sierra del Cristal, lower Río Levisa, gallery forest, serpentine

Pleurothallis grisebachiana Cogn.

Stenzel 600

CU

Ho: Sierra de Moa, Toldo Massif, road to Moa, gallery forest, serpentine

Pleurothallis grisebachiana Cogn.

Stenzel 605

CU

SS: Sierra de Escambray, Pico de Potrerillo, summit, limestone

Pleurothallis grisebachiana Cogn.

Stenzel 612

CU

SS: Sierra de Escambray, Cascada de Vegas Grandes, limestone

Pleurothallis grisebachiana Cogn.

Stenzel 619

CU

Gr: Sierra Maestra, Victorino, mogote at road, limestone

Pleurothallis grisebachiana Cogn.

Stenzel 865

CU

Gu: Yateras, Pico Galán, N slope, gallery forest, serpentine

Pleurothallis grisebachiana Cogn.

Stenzel 882

CU

Gu: Sierra de Imías, La Yamagua, charrascal along creek, serpentine

Pleurothallis grobyi Batem. ex. Lindl.

G. Vargas & al. 1543

CR

Puntarenas, Cantón de Osa

Pleurothallis haitiensis D.D.Dod

ex NY 9207

HA

 

Pleurothallis helenae Fawc. & Rendle

Stenzel 637

CU

Gr: Sierra Maestra, Victorino, El Gigante, summit

Pleurothallis hirsutula Fawc. & Rendle

C. Whitefoord 1394 ex BM 82345

JA

Trelawny, on damp rock outside Windside Cave

Pleurothallis lanceola (Sw.) Lindl.

Maxon 9536 ex NY 59959

JA

St. Thomas, Maccasucker Bump

Pleurothallis laxa (Sw.) Lindl.

Morris s.n. ex K

JA

Blue Mountains

Pleurothallis llamachoi Luer

Stenzel 583

CU

Ho: Sierra de Moa, Meseta del Toldo, road to Moa, gallery forest at creek

Pleurothallis longilabris Lindl.

Stenzel 895

CU

Gu: Sierra de Imías, La Yamagua, centre of valley above creek

Pleurothallis miguelii Schltr.

ex NY 59972

  

Pleurothallis mitchellii D.D.Dod

ex NY 9209

  

Pleurothallis mucronata Lindl. ex. Cogn.

Stenzel 478

CU

Ho: Sierra de Nipe, Cayo Las Mujeres, W slope, broadleafed dwarf forest

Pleurothallis mucronata Lindl. ex. Cogn.

Stenzel 520

CU

Ho: Sierra del Cristal, S slope, along upper Río Levisa

Pleurothallis mucronata Lindl. ex. Cogn.

Stenzel 560

CU

Ho: Sierra de Moa, Meseta del Toldo, along headwaters of Río Piloto

Pleurothallis murex Rchb. f.

Martinez-Falcon s.n. ex HAC 41200

CU

SS: Yaguajay, Lomas de Canoa

Pleurothallis obliquipetala Acuña & C. Schweinf.

Stenzel 653

CU

Gr: Sierra Maestra, Turquino Massif, below La Aguada de Joaquín

Pleurothallis obovata (Lindl.) Lindl.

Stenzel 692

CU

?

Pleurothallis odontotepala Rchb. f.

Stenzel 67

CU

Gr: Sierra Maestra, Turquino Massif, Alto de Naranjo (Pico Mella)

Pleurothallis oricola H.Stenzel

Stenzel 1210

CU

PR: Guanahacabibes, trail from María la Gorda to Cabo Corrientes

Pleurothallis papulifolia Luer

Stenzel 483

CU

Ho: Sierra de Nipe, Woodfred, along N side of creek

Pleurothallis prostrata Lindl.

Stenzel 581

CU

Ho: Sierra de Moa, Meseta del Toldo, unpaved road to Moa, gallery forest along a dry creek

Pleurothallis pruinosa Lindl.

Splitgerber 527

SU

?

Pleurothallis pruinosa Lindl.

Herrera 6330 ex K

CR

Alajuela, Bijagua

Pleurothallis pubescens Lindl.

Ackerman 2041 ex UPRRP 6289

PR

Mun. Utuado, Cerro Morales

Pleurothallis quisqueana D.D.Dod

Alain 15483 ex NY 59544

DR

 

Pleurothallis quisqueana D.D.Dod

ex NY 59709

  

Pleurothallis aff. racemiflora (Sw.) Lindl.

Stenzel 888

CU

Gu: Sierra de Imías, La Yamagua, charrascal and gallery forest along creek on serpentine

Pleurothallis racemiflora (Sw.) Lindl.

Stenzel 643

CU

Gr: Sierra Maestra, Turquino Massif, below La Aguada de Joaquín, Rascacielo

Pleurothallis rubroviridis Lindl.

Stenzel 880

CU

Gu: Sierra de Imías, Los Calderos, ridge E from the valley

Pleurothallis ruscifolia (Jacq.) R. Br.

s.n.

?

Botanical Garden Munich

Pleurothallis ruscifolia (Jacq.) R. Br.

C.K.Horich s.n. (Univ. Calif. B.G. Berkeley acc# 59.270-1) ex K

CR

Cerro Cedral

Pleurothallis ruscifolia (Jacq.) R. Br.

ex NY 59721

?

 

Pleurothallis ruscifolia (Jacq.) R. Br.

ex NY 59720

  

Pleurothallis ruscifolia (Jacq.) R. Br.

ex NY 59783

  

Pleurothallis sertularioides (Sw.) Spreng.

Stenzel 603

CU

SS: Sierra de Escambray, Pico de Potrerillo, summit

Pleurothallis shaferi Ames

Stenzel 453

CU

Ho: Sierra de Nipe, La Mensura, N slope

Pleurothallis simpliciflora D.D.Dod

ex NY 9215

  

Pleurothallis testaefolia (Sw.) Lindl.

Stenzel 75

CU

SS: Sierra Maestra, Turquino Massif, below La Aguada de Joaquin

Pleurothallis tribuloides (Sw.) Lindl.

Stenzel 634

CU

Gu: Sierra Maestra, Victorino, El Gigante, Barril forest at summit

Pleurothallis trichophora Lindl.

Wright 659 ex K-L

CU

E Cuba

Pleurothallis trichyphis Rchb. f.

Stenzel 620

CU

Gr: Sierra Maestra, Victorino, mogote at road

Pleurothallis velaticaulis Rchb. f.

Fendler 1472 ex K-L

VE

?

Pleurothallis velaticaulis Rchb. f.

L. Williams & A.H.G. Alston 300 ex BM 82425

VE

El Avila

Pleurothallis wilsonii Lindl.

Stenzel 845

CU

Gu: Palenque, Piedra La Vela, gallery forest along Arroyo Sonador

Pleurothallis wrightii Rchb. f.

Stenzel 442

CU

Ho: Sierra de Nipe, La Mensura, N slope

Stelis pygmaea Cogn.

Stenzel 669

CU

SC: Sierra Maestra, Santiago de Cuba, La Gran Piedra

Trichosalpinx dura (Lindl.) Luer

PFC #66454 ex HAJB

CU

SS: Sierra de Escambray, Pico de Potrerillo

Zootrophion atropurpureum (Lindl.) Luer

s.n.

?

living collection I. Bock, Naumburg

Zootrophion atropurpureum (Lindl.) Luer

Stenzel 811

CU

Gr: Sierra Maestra, Turquino Massif, between Loma de León and La Aguada de Joaquín

* – CR = Costa Rica, CU = Cuba, DR = Dominican Republic, EC = Ecuador, HA = Haiti, JA = Jamaica, NI = Nicaragua, PR = Puerto Rico, SU = Surinam, VE = Venezuela. ** – Gr = Prov. Granma, Gu = Prov. Guantánamo, Ho = Prov. Holguin, PR = Prov. Pinar del Río, SC = Prov. Santiago de Cuba, SS = Prov. Santi Spíritus.

[page 23↓]Genetics

Plant material used in this study is listed in Tab. 3. All but 2 of the 39 Pleurothallis species (Pleurothallis appendiculata, P. murex) currently known to occur in Cuba could be included, i.e. the range is limited geographically. One of the 33 species successfully sampled could not be recollected in Cuba, i.e. material from other islands had to be employed (Pleurothallis aristata from Puerto Rico). Two species are represented by two accessions each. P. trichophora with material from East and Central Cuba which marks the phytogeographical limits of this plant, and P. ghiesbreghtiana with one voucher from West Cuba and the other from Puerto Rico.

Tab. 3: Plant materials used in molecular studies.

Species

Voucher

Country* & Locality**

Pleurothallis aristata Hooker

Stenzel 996

PR

Mun. Río Grande, El Yunque, trail from Caimitillos to Mt. Britton

Pleurothallis bissei Luer

Stenzel 730

CU

Ho: Sierra de Nipe, Woodfred, along N bank of creek

Pleurothallis brighamii S. Wats.

Stenzel 740

CU

Ho: Sierra de Nipe, behind EIIM station and Sabina trail

Pleurothallis corniculata (Sw.) Lindl.

Stenzel 889

CU

Gu: Sierra de Imías, La Yamagua

Pleurothallis domingensis Cogn.

Stenzel 662

CU

SC: Sierra Maestra, Turquino Massif, Pico Real

Pleurothallis ekmanii Schltr.

Stenzel 762

CU

Ho: Sierra del Cristal, S slope of Pico Cristal, trail to summit

Pleurothallis excentrica (Luer) Luer

Stenzel 752

CU

Ho: Sierra del Cristal, S slope of Pico Cristal, along upper Río Levisa

Pleurothallisflabelliformis’ H. Stenzel

Stenzel 745

CU

Ho: Sierra del Cristal, S slope of Pico Cristal, along upper Río Levisa

Pleurothallis gemina H. Stenzel

Stenzel 452

CU

Ho: Sierra de Nipe, La Mensura, north slope

Pleurothallis ghiesbreghtiana A. Rich. & Galeotti

Stenzel 967

PR

Maricao, Bo. Maricao Afuera, Maricao Forest Reserve

Pleurothallis ghiesbreghtiana A. Rich. & Galeotti

Stenzel 1298

CU

PR: Pan de Guajaibón, summit

Pleurothallis grisebachiana Cogn.

Stenzel 619

CU

Gr: Sierra Maestra, Victorino, mogote at road

Pleurothallis helenae Fawc. & Rendle

Stenzel 766

CU

Ho: Sierra del Cristal, Pico Cristal, elfin forest on peak

Pleurothallis llamachoi Luer

Stenzel 545

CU

Ho: Sierra del Cristal, lower Río Levisa, gallery forest

Pleurothallis longilabris Lindl.

Stenzel 895

CU

Gu: Sierra de Imías, La Yamagua, centre of valley above creek

Pleurothallis mucronata Lindl. ex. Cogn.

Stenzel 478

CU

Ho: Sierra de Nipe, Cayo Las Mujeres, W slope

Pleurothallis nummularia Rchb. f.

Stenzel 896

CU

Gu: Sierra de Imías, La Yamagua, centre of valley above creek

Pleurothallis obliquipetala Acuña & C. Schweinf.

Stenzel 789

CU

Gr: Sierra Maestra, Turquino Massif, below La Aguada de Joaquín

Pleurothallis obovata (Lindl.) Lindl.

Stenzel 840

CU

Gu: Palenque, Mogote de Buena Vista, along crest

Pleurothallis odontotepala Rchb. f.

Stenzel 784

CU

Gr: Turquino Massif, trail from Alto de Naranjo to La Aguada de Joaquín

Pleurothallis papulifolia Luer

Stenzel 483

CU

Ho: Sierra de Nipe, Woodfred, along N side of creek

Pleurothallis prostrata Lindl.

Stenzel 856

CU

Gu: Yateras, W slope of Pico Galán

Pleurothallis pruinosa Lindl.

Stenzel 890

CU

Gu: Sierra de Imías, La Yamagua, Monte Oscuro

Pleurothallis racemiflora (Sw.) Lindl.

Stenzel 783

CU

Gr: Sierra Maestra, Turquino Massif, below La Aguada de Joaquín

Pleurothallis rubroviridis Lindl.

Stenzel 893

CU

Gu: Guantánamo, Sierra de Imías, La Yamagua

Pleurothallis ruscifolia (Jacq.) R. Br.

Stenzel 635

CU

Gr: Sierra Maestra, Bayamo, Victorino, peak of El Gigante

Pleurothallis sertularioides (Sw.) Spreng.

Stenzel 843

CU

Gu: Palenque, Mogote de Buena Vista, along crest

Pleurothallis shaferi Ames

Stenzel 453

CU

Ho: Sierra de Nipe, La Mensura, N slope

Pleurothallis testaefolia (Sw.) Lindl.

Stenzel 656

CU

SS: Sierra Maestra, Turquino Massif, below La Aguada de Joaquin

Pleurothallis tribuloides (Sw.) Lindl.

Stenzel 634

CU

Gu: Sierra Maestra, Victorino, El Gigante

Pleurothallis trichophora Lindl.

Stenzel 606

CU

SS: Trinidad, Pico Potrerillo

Pleurothallis trichophora Lindl.

Stenzel 630

CU

Gu: Sierra Maestra, Victorino, El Gigante

Pleurothallis trichyphis Rchb. f.

Stenzel 620

CU

Gr: Sierra Maestra, Victorino, mogote at road

Pleurothallis wilsonii Lindl.

Stenzel 621

CU

Gr: Sierra Maestra, Victorino, mogote at road

Pleurothallis wrightii Rchb. f.

Stenzel 733

CU

Ho: Sierra de Nipe, Cayo Las Mujeres, W slope

* – CU = Cuba, HA = Haiti, PR = Puerto Rico. ** – Gr = Prov. Granma, Gu = Prov. Guantánamo, Ho = Prov. Holguin, PR = Prov. Pinar del Río, SC = Prov. Santiago de Cuba, SS – Prov. Sancti Spíritus.


[page 24↓]

Material – Since it was not possible to extract DNA from existing herbarium collections, material for molecular investigations had to be collected in the wild. Outside ITS sequences for outgroup comparison were kindly provided by Dr. A. M. Pridgeon. They were originally published by Pridgeon & al. (2001) and will be cited hereafter, if necessary, under the GenBank accession number.

DNA extraction – DNA was extracted at Friedrich-Schiller-University in Jena from 0.01-0.5g of dried leaves or fruits, following a modified 2%-CTAB (hexadecyltri-methylammoniumbromide) procedure of Doyle & Doyle (1987) and Hellwig & al. (1999). DNA was purified using Qiagen tip-20 columns (Qiagen Inc., Hilden, Germany) following the manufacturer’s protocols. Purified DNA extracts are stored in HUB.

Amplification – All PCRs were run using Taq DNA Polymerase Kit from Qiagen (Qiagen Inc.). Initially, Baldwin’s (1992) primers P1 (=”ITS5” sensu Baldwin), P2 (=”ITS2”), P3 (=”ITS3”), P4 (=”ITS4”) were employed. They yielded two fragments. One was the result of primers P1 (5'-GGA AGT AAA AGT CGT AAC AAG G-3') and P2 (5'-CTC GAT GGA ACA CGG GAT TCT GC-'3). It covered ITS1 and 2/3 of 5.8S gene with a total length of ~380 bp. The other fragment of ~440 bp included 2/3 of the 5.8S gene and ITS2. It was the product of primers P3 (5'-GCA TCG ATG AAG AAC GCA GC-'3) and P4 (5'-TCCTCCGCTTATTGATATGC-'3). Both fragments are overlapping in the 5.8S region, so by combining the two, the complete ITS1-5.8S-ITS2 (hereafter ITS) sequence could be achieved.

Tab. 4: Pleurothallis gemina: 17SE (p.p.), ITS1, 5,8S, ITS2, and 26SE (p.p.) sequences.

TGGAAGTAAAAGTCGTAACAAGG>TTTCCGTAGGTGAACCTGCGGAAGGATCATTGtcgagaccgaatatatcgagcgattcggagaacctgtgaatgaatgagcggcggcatcgtcgtcgcgcaaatgccgcctccatcgccggccttgttaaagcgccgtcgatgagaggcggatgaaaactcaaaccggcgcagctgcgcgccaaggaaatacaaatatacacgatcctgtattgggttcggtggcgtggagtgcagtcgcacaccacacggataAAAACGACTCTCGGCAATGGATATCTCGGCTCTCGCATCGATGAAGAGC G CAGC>GAAATGCGATACGTGGTGCGAATT<GCAGAATCCCG C G AA CCATCGAGAATTTGAACGCAAGTTGCGCCCGAGGCCAGCCGGCCAAGGGCACGTCCGCCTGGGCGTCAAGCgttacgtcgctccgtgccagctccatgccacccgacgggtgtgtatggggaggggtcggatgtgcagagtggctcgtcgtgcccgcgggcgtggcgggcttaagatcgggtgatcgtctcgtttgccacgaacgataagggtggatgaaaattgtgcctgtgttgtatcgtggcgacgtgagaagagattgtacccagcagatgatcccaatctaagcgtcgttccacagacgtcggcttggaatGCGACCCCAGGATGGGCGAGGCCACCCGCTGAGTTTAA<GCATATCAATAAGCGGAGGAAAGAAACTTACAAG

Noncoding (ITS) regions in lower case, primer target regions underlined, primer pair P1/P2 in red, primer pair P3/P4 in blue, amplification direction of primers indicated by „<“ and „>“. Base mismatches in primers in bold type.

Tab. 4 illustrates the position of each primer within the nrDNA region of interest. Baldwin’s primers gave ad hoc excellent results in ITS1 (P1-P2) but less so in the case of ITS2 (P3-P4). Adjusting of the annealing temperature did not improve the quantity and quality of the products. Since the PCR with P1-P4 gave the same blurred PCR products as P3-P4, I [page 25↓]assumed that the P4 must include bases mismatching the 26SE target region. However, this cannot explain the notorious difficulty amplifying ITS2. Rather than base mismatches, secondary structures could explain those problems, for later usage of P4 in nested cycle sequencing with templates gained from non-P1-P4 PCR, showed no problems at all.
Sequence analyses revealed a three base mismatch in primer P2 compared with the actual 5.8S target region in all species examined, whereas primer P3 has one base mismatch (Tab. 4). Cycle sequencing functioned well despite these incongruities.

In those cases where PCR with Baldwin’s primers failed, a second set of oligos was employed (Sun & al. 1994). Using the primers “17SE” (5'-ACG AAT TCA TGG TCC GGT GAA GTG TTC G-3') and “26SE” (5'-TAG AAT TCC CCG GTT CGC TCG CCG TTA C-3') PCR yielded a fragment of ~ 900 bp comprising ITS1, 5.8S gene, and ITS2 along with ends of the bordering coding regions. Since these oligos anneal further outwards from primers P1 and P4, P1-P4 could be used for nested cycle sequencing.
The amplification was conducted via the following thermal cycle profile: 150s95°C/ 28-34x [30s95°C/60s50-58°C/ 60s72°C]/ 300s72°/ 4°C. PCR products were purified using the QiaQuick® PCR Purification Kit (Qiagen Inc.).

Cycle sequencing – Both strands of the amplified gene fragments were directly cycle sequenced in 10 µl volumes containing 2 µl of ABI Prism BigDye Terminator cycle sequencing reaction mix (ABI Biosys-tems), 0.5 µM primer, 2 µl dd H2O and 4 µl DNA. Sequencing products were purified following the ABI standard protocol adjusted to 10 µl reaction volume by addition of dd H2O. Sequences were obtained by running the sequencing products on an Perkin Elmer ABI 377 automated sequencer. The resulting sequence electropherograms of both strands were corrected manually for misreads and merged into one sequence file using BioEdit (version 5.0.9.; Hall 1999) for Windows 95, which was generally used to store and manage sequences.

Alignment and phylogenetic analysis – The question of homology is a crucial problem in inferring phylogenetic relationships from nucleotide sequence data (Doyle & Davis 1998), i.e. the insertion of gaps is one of the most critical steps in molecular analysis. To compare the influence of the extent of indels, sequences were initially aligned using CLUSTAL W (Thompson & al. 1994) employing different delay values and gap costs and then adjusted by eye (see appendix for sequences). Unambiguous indels that seem to be duplications of upstream sequences (e.g. ATAT à ATATAT) were coded as single events. From each matrix an additional set with ambiguous indel regions being excluded was drawn. A fifth matrix was created by stripping an aligned set of all columns containing gaps. Cladistic analysis was conducted with these 5 data sets using sequence AF262915 (Dilomilis montana (Sw.) Summerh.) from (Pridgeon & al. 2001) as outgroup.
Substitution patterns were analysed by comparing observed and expected substitutions [page 26↓]among all sequences. The number of expected substitutions was calculated from base frequencies. Substitution saturation was evaluated by plotting portions of transitions and transversion against distance.
Phylogenetic trees were built using parsimony algorithms (Maximum Parsimony – MP hereafter). Computation was accomplished with PAUP* version 4.0b8 (Swofford 2000). The following settings were chosen: Fitch parsimony (Fitch 1971), heuristic search, gaps as fifth character, 100 replicates of random sequence addition, tree bisection-reconnection (TBR) branch swapping, MulTree.

Relative support of the topologies found was evaluated with 1000 bootstrap subsets of each of the 5 matrices.

Sequence editing and statistical analysis were performed using BioEdit and DAMBE (version 4.1.19; Xia & Xie 2001).

Taxonomy

Although profoundly challenged by molecular data (Pridgeon, Solano & Chase 2001, Pridgeon & Chase 2001) the morphological concept of Luer (1986c) will be the basis of this work. The main reasons are

  1. less than 60 % of the supraspecific taxa proposed by Luer and included in that molecular study were represented by the type species.
  2. Pridgeon & Chase (2001) based their ~500 nomenclatural transfers on the sequences of 187 pleurothallid specimens. Those epithets not sequenced were transferred merely on the basis of Luer’s morphological system. A great number of taxa of Luer’s system, however, had been shown to be paraphyletic in the same molecular study.
  3. 15 of the 39 Cuban species were transferred to other genera, however, only 3 species were effectively included in the molecular screening.

There are more reasons why to handle the new system with extreme care. In order not to anticipate the results of this work they will be discussed later (p. 112).

General rules

Authority abbreviations follow those of Brummit & Powell (1992) and abbreviations of herbaria are in concordance with those of the Index Herbariorum (http://www.nybg.org/bsci/ih/searchih.html). Name of the Cuban provinces were abbreviated according to the rules of the FCP (Greuter & al. 2002): PR – Pinar del Río, SS – Santi Spíritus, Ci – Cienfuegos, Ho – Holguín, Gr – Gránma, SC – Santiago de Cuba, Gu – Guantánamo.


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