High genetic variability in self-incompatible myophilous Octomeria (Orchidaceae, Pleurothallidinae) species

REPRODUCTIVE BIOLOGY

High genetic variability in self-incompatible myophilousOctomeria (Orchidaceae, Pleurothallidinae) species

Ariane Raquel Barbosa • Viviane Silva-Pereira •

Eduardo Leite Borba

Received: 23 October 2012 / Accepted: 19 September 2013 / Published online: 31 October 2013

� Botanical Society of Sao Paulo 2013

Abstract Octomeria crassifolia Lindl. and O. grandiflora

Lindl. are myophilous, self-incompatible and partially

inter-compatible species. In order to better understand the

relationships between their reproductive systems, patterns

of genetic variability, and isolation mechanisms in sym-

patric populations, a genetic study using ISSR molecular

markers was carried out in natural populations growing in

southeastern Brazil. The populations of both species

demonstrated moderately high genetic variability greater

than that observed in other self-compatible melittophilous

orchid species, indicating that self-incompatibility may be

a determinant factor in maintaining higher variability levels

in myophilous Pleurothallidinae species. Contrary to what

might be expected due to behavior of their pollinators of

flying short distances, these species of Octomeria demon-

strated relatively low genetic structuring that was probably

related to gene flow by seeds or to older shared genetic

stock. Bayesian analysis of genetic structuring indicated

the presence of three genetic groups in O. crassifolia

(although without any relation to their geographical dis-

tributions) and two genetic groups in O. grandiflora (with

one of them restricted to one of the populations). No

indications were seen of hybridization or introgression

among the sympatric populations, indicating that pollinator

specificity is an important factor in guaranteeing the

identities of these inter-compatible species.

Keywords Isolation mechanisms � ISSR �Myophily �Population genetics � Self-incompatibility

Introduction

Genetic variability in plant populations is influenced by

many factors, including their modes of reproduction, seed

dispersal mechanisms, geographical distributions, popula-

tion sizes, lifecycles, and life forms, and especially their

mating systems (Loveless and Hamrick 1984; Nybom and

Bartish 2000). Species that preferentially reproduce by

allogamy usually demonstrate moderate to elevated levels

of genetic variation within their populations, with the

levels of differentiation between those populations gener-

ally being low in comparison to autogamous species

(Hamrick and Godt 1990; Sun and Wong 2001). Most

Orchidaceae are self-compatible, but pre-pollination bar-

riers help avoiding self-pollination and maintaining genetic

variability (van der Pijl and Dodson 1966; Dressler 1981;

Borba and Semir 1999; Singer and Cocucci 1999; Borba

et al. 2007; Smidt et al. 2006; Silva-Pereira et al. 2007;

Cruz et al. 2011). Pollinator behavior also influences

genetic variability in plant populations. Dipterans, which

pollinate almost all species of the subtribe Pleurothallidi-

nae (ca. 4,100 spp.) and the genus Bulbophyllum (ca. 1,200

spp.), exhibit behaviors that favor self-pollination as they

tend to make long visits to numerous flowers of the same

inflorescence or of the same individual (Borba and Semir

A. R. Barbosa

Departamento de Botanica, Instituto de Ciencias Biologicas,

Universidade Federal de Minas Gerais, Av. Antonio Carlos,

6627, Pampulha, Belo Horizonte, MG 31270-901, Brazil

V. Silva-Pereira

Setor de Ciencias Biologicas, Departamento de Botanica,

Universidade Federal do Parana, Centro Politecnico, Jardim das

Americas, Curitiba, PR 81531-990, Brazil

E. L. Borba (&)

Centro de Ciencias Naturais e Humanas, Universidade Federal

do ABC, Rua Santa Adelia, 166, Bangu, Santo Andre,

SP 09210-170, Brazil

e-mail: [email protected]

123

Braz. J. Bot (2013) 36(3):179–187

DOI 10.1007/s40415-013-0027-0

1998, 2001). As such, reduced genetic diversity might be

expected to be found among populations of myophilous

(pollinated by dipterans) orchids. However, populations of

Acianthera (Pleurothallidinae) species, a myophilous and

self-incompatible genus, demonstrate high genetic vari-

ability as compared to other Orchidaceae species (Borba

et al. 2001a, 2001b). Borba et al. (2011) suggested that the

maintenance of high levels of genetic diversity in these

populations has been possible due to the occurrence of self-

incompatibility, a characteristic that may have arisen in

response to pollinator behavior.

The genus Octomeria, an early divergent genus in the

myophilous clade of the subtribe Pleurothallidinae (Prid-

geon et al. 2001), comprises approximately 150 Neotropi-

cal species, concentrated mainly in the northern region of

South America and in southeastern Brazil (Luer 1986).

Octomeria crassifolia Lindl. is widely distributed in Minas

Gerais State, and its taxonomic delimitation was recently

expanded as its populations demonstrated wide morpho-

logical and floral variations that overlapped with what were

previously considered to be distinct species (Forster 2007).

The morphological amplitude of O. crassifolia may be

related to its adaptations to different phytophysiognomies,

occurring as an epiphyte in gallery or humid forests or with

a rupicolous habit on granitic, quartzitic, or ferruginous

outcrops. The taxonomic delimitation of Octomeria gran-

diflora Lindl. was also revised recently. This species is

widely distributed in southeastern Brazil, but is principally

found in gallery forests along watercourses (Forster 2007).

In contrast to O. crassifolia, populations of O. grandiflora

differ among themselves only in terms of the sizes of their

flowers and ramicauls and the colors of their perianth

(which can vary from light to intense yellow) (Forster

2007).

Experimental pollinations in populations of four species

of Octomeria demonstrated the occurrence of self-incom-

patibility in the genus, with the total absence of fruit set in

self-pollinations in O. crassifolia and O. grandiflora

(Barbosa et al. 2009; Borba et al. 2011). These species,

which are occasionally sympatric and flower in partial

synchrony, are inter-compatible, although interspecific fruit

set is low. Because of this, these species could hybridize in

nature unless they had efficient pre-pollination barriers.

Barbosa et al. (2009) noted that the main pollinators of O.

crassifolia and O. grandiflora were species of Bradysia and

Pseudosciara (Diptera, Sciaridae), respectively, insects

with different body sizes and presenting high specificity

that makes interspecific pollination in sympatric popula-

tions more difficult, in spite of similarities in their floral

characteristics. Nonetheless, due to the frequently promis-

cuous behavior of many species of dipterans (van der Pijl

and Dodson 1966), hybridization could occur if this insect-

plant fidelity was compromised.

In this study, we sought to determine the genetic

structures and variability in Octomeria crassifolia and O.

grandiflora populations in order to verify if: (1) the species

and populations present high genetic variability, as

observed in other myophilous self-incompatible orchid

species (Borba et al. 2001b; Azevedo et al. 2007; Ribeiro

et al. 2008); (2) the species present moderate to high

genetic structuring, as found in some other myophilous

orchid species possibly due to the behavior of their flies

pollinators fly short distances (Borba et al. 2001b; Ribeiro

et al. 2008); and (3) there is evidence of natural hybrid-

ization between sympatric populations of the two species,

which would be avoided by the pollinator specificity

(Barbosa et al. 2009).

Materials and methods

Plant material

We sampled leaves of individuals from four populations of

Octomeria crassifolia in Minas Gerais state. A large mor-

phological variation was observed within and among the

populations, and the plants collected could grow as epi-

phytes in gallery forest or rupicolous in granite, quartzite

and ferruginous outcrops (Fig. 1; Table 1). For O. gran-

diflora, we collected leaves of individuals from three

populations in Minas Gerais and one from Rio de Janeiro

state. The habitat and morphological characteristics in the

populations of O. grandiflora were apparently more

homogeneous than those of O. crassifolia (Fig. 1; Table 1).

Populations of both species were sympatric in the Serra do

Caraca, at Catas Altas municipality in Minas Gerais state.

DNA isolation and ISSR-PCR

Approximately 100 mg of dry leaves were used for DNA

extraction according to Doyle and Doyle (1987) protocol.

The cetyltrimethylammonium bromide (CTAB) protocol

uses the following buffer: 100 mM Tris pH 8.0, 20 mM

ethylenediamine tetraacetic acid (EDTA), 1.4 M NaCl,

2 % CTAB, 1 % polyvinylpyrolidone (PVP), and 2 % b-

mercaptoethanol. The concentration of DNA was visually

quantified using 0.8 % agarose gel by comparison with

standard DNA concentrations. DNA was diluted in double-

distilled water to a final concentration of approximately

20 ng/lL prior to PCR amplifications.

PCR amplifications were carried out in a total volume of

20 lL, containing 2.0 lL 109 PCR buffer (including

1.5 mM MgCl2), 0.20 mM dNTPs, 0.32 lM primer, 0.5

unit Taq polymerase (Phoneutria), and 20 ng DNA tem-

plate. The program consisted of an initial denaturation at

94 �C for 4 min, followed by 37 cycles of 1 min at 94 �C,

180 A. R. Barbosa et al.

123

2 min at 46, 47.6, or 50 �C depending on the primer

(Table 2), 2 min at 72 �C and a final extension of 7 min at

72 8C. Amplification products were electrophoretically

separated at a constant voltage of 60 V for 4 h in 1.5 %

agarose gels with 0.59 TAE buffer, stained with ethidium

bromide and photographed under UV light. A 100-bp DNA

ladder was used to estimate the molecular size of the

fragments. Thirty-one primers were tested to identify those

that produce reproducible markers. Eight primers were

selected for use in the amplification of 65 individuals of

Octomeria crassifolia and 54 individuals of O. grandiflora.

Six coincident primers for both species were selected for

sympatric populations (Table 2). The replicability of the

primers was tested by redoing several individuals.

Data analyses

The phenotypic profile of all individuals for each primer was

determined through the comparative analysis of images of

agarose gels. The fragments amplified were scored as 1

(presence) or 0 (absence) and converted into a binary data

matrix. The software GenAlEx 6.0 (Peakall and Smouse

2006) was used to obtain estimates of genetic diversity

parameters for each species: total number of loci in each

population (N), mean Shannon’s index of phenotypic

diversity (I), percentage of polymorphic loci (P), and mean

expected heterozygosity (HE-pop). The expected heterozy-

gosity values for the species were calculated with the soft-

ware PopGene v. 1.32 (Yeh et al. 1997). A Principal

Coordinate Analysis (PCO) was carried out using the matrix

of Nei’s genetic distance (1978), using Genealex 6. Three

Analyses of Molecular Variance (AMOVAs) were per-

formed in Genealex 6, two considering all populations of

each species, and one for the two sympatric populations. The

Bayesian algorithm in AFLP-SURV (Vekemans 2002) was

used to generate 1,000 dissimilarity matrices with Nei’s

genetic distance and the FST values, which were used to

construct 1,000 neighbor-joining trees using the NEIGH-

BOR module in PHYLIP 3.69 (Felsenstein 2006). Bootstrap

values were obtained computing a majority-rule consensus

tree at the CONSENSE module in PHYLIP. Trees obtained

were visualized and edited in FigTree 1.2 (Rambaut 2008).

Three Bayesian structure analyses were performed using

the software STRUCTURE 2.2 (Pritchard et al. 2000) to

infer the number of genetic clusters (K) in each species and

for sympatric populations. The number of presumed pop-

ulations (K) was set of K = 1 to K = 5 for each species

and of K = 1 to K = 5 for the two sympatric populations.

Ten independent runs were performed for each K. Each run

was pursued for 500,000 Markov Chain Monte Carlo

(MCMC) iterations, with an initial burn-in of 100,000

iterations, with the admixture model with alleles correlated

among populations. To infer the number of genetic clus-

ters, we calculated the average of each K likelihood value,

‘‘log of probability’’ (LnP(D)), through all runs as sug-

gested by Pritchard et al. (2000) and the statistic Delta

K according to Evanno et al. (2005) with STRUCTURE

HARVESTER 0.6.8 (Earl and von Holdt 2012).

Fig. 1 Distribution of

Octomeria crassifolia and O.

grandiflora (Orchidaceae)

populations sampled. For

population names see Table 1

Genetic variability of Octomeria (Orchidaceae) species 181

123

Results

Octomeria crassifolia

Oc-NEG showed the smallest number of fragments per

population (81), while Oc-PIE (89) showed the largestTa

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2 Table 2 Primers used for ISSR amplification of Octomeria crassi-

folia, O. grandiflora (Orchidaceae) populations, and two sympatric

populations of both species

Primer

name

Primer

sequenceaO.

crassifolia

O.

grandiflora

Sympatric

populations

T �C N

loci

T �C N

loci

T

�C

N

loci

AW3 (GT)6RG 50 9 – – – –

DAT (GA)7RG – – 50 13 – –

JOHN (AG)7YC 50 11 50 15 50 21

MANNY (CAC)4RC 46 14 47.6 16 47.6 14

MAO (CTC)4RC 50 12 50 16 50 20

UBC 840 (GA)8YT 47.6 15 46 8 47.6 22

UBC 880 (GGAGA)3 47.6 13 46 9 47.6 17

UBC 898 (CA)6RY 46 10 – – – –

UBC 899 (CA)6RG 46 8 47.6 14 47.6 14

UBC 901 (GT)6YR – – 47.6 7 – –

TOTAL 92 98 108

T �C annealing temperature, N loci number of loci analyzed for each

primera Y = C or T; R = A or G

Table 3 Estimates of genetic diversity parameters of four popula-

tions of Octomeria crassifolia and four populations of O. grandiflora

(Orchidaceae), based on 92 and 98 ISSR loci, respectively

Species/population N I P (%) HE

O. crassifolia

Oc-CAR 87 0.404 (0.022) 92.39 0.267 (0.017)

Oc-PIE 89 0.392 (0.022) 93.48 0.256 (0.017)

Oc-PAP 88 0.406 (0.021) 93.48 0.265 (0.016)

Oc-NEG 81 0.400 (0.023) 86.96 0.271 (0.018)

Species – 0.530 (0.133) – 0.352 (0.114)

O. grandiflora

Og-CAR 83 0.402 (0.025) 80.61 0.279 (0.019)

Og-BRI 91 0.420 (0.023) 87.76 0.283 (0.018)

Og-MAC 87 0.353 (0.023) 84.69 0.231 (0.017)

Og-AMB 84 0.353 (0.025) 76.53 0.238 (0.019)

Species – 0.508 (0.171) – 0.338 (0.138)

N total number of loci in each population, I mean Shannon’s index of

diversity, P percentage of polymorphic loci, HE mean expected

heterozygosity

Standard deviation in parentheses

For population names see Table 1


123

(Table 3). None of the populations showed exclusive

fragments. Shannon Index values varied from 0.392 (Oc-

PIE) to 0.406 (Oc-PAP). The proportions of polymorphic

loci in the populations varied from 86.96 % (Oc-NEG) to

93.48 % (Oc-CAR and Oc-PAP). The expected heterozy-

gosity of this species was 0.352, with Oc-PIE having the

smallest HE value (0.256) and Oc-NEG the largest (0.271)

(Table 3). Interpopulational variability estimated by ana-

lysis of molecular variance (AMOVA) demonstrated high

variation within the populations (92 %) and low diver-

gence among them (8 %).

The genetic structuring value calculated by UST was

0.076, and the dendrogram constructed using Nei’s genetic

distance did not demonstrate existence of clear subgroups

among the four populations. There was, however, a linkage

presenting between the populations Oc-NEG and Oc-PAP

(0.020) and subsequent chaining of the other populations,

with Oc-PAP and Oc-PIE showing the largest genetic dis-

tance (0.048) (Fig. 2a). The first two axes explained

43.01 % of the variation in the Principal Coordinate Ana-

lysis (PCO) and corroborated the clustering analysis, indi-

cating a structure in gradient with partial to almost complete

overlapping of the populations (Fig. 3a). This same pattern

was observed in Bayesian structure analysis, with the

average probability values LnP(K) and DK indicating three

distinct genetic groups, but no population presented an

exclusive genetic pool (Fig. 4a). All the populations pre-

sented a mix of the three genetic pools in different pro-

portions, corroborating the structuring scene in PCO.

Octomeria grandiflora

Og-CAR population had the smallest number of fragments

(83), while the largest number was found in population Og-

BRI (91) (Table 3). The polymorphism values were lower

than those found for O. crassifolia, with the lowest value

found in Og-AMB (76.53 %) and the highest in Og-BRI

(87.76 %). The expected heterozygosity for the species was

0.338, with the smallest value found in Og-MAC (0.231)

Fig. 2 Neighbor-joining dendrogram showing the phenetic relation-

ships among a four populations of O. crassifolia (based on 92 ISSR

loci); b four populations of O. grandiflora (based on 98 ISSR loci),

constructed using the matrix of genetic distances (Nei 1978; unbiased

estimate). Bootstrap percentages (50 % or more) are presented in the

branches for FST (above) and genetic distances (below). For


Fig. 3 Representation of the scores on the first two axes of the

principal coordinate analysis (PCO) from the matrix of genetic

distances based on ISSR loci of individuals from a four populations of

Octomeria crassifolia (axis 1 = 23.68 % and axis 2 = 19.34 % of

variance); b four populations of O. grandiflora (axis 1 = 26.35 %

and axis 2 = 17.50 % of variance); c sympatric populations of both

species (axis 1 = 29.47 % and axis 2 = 22.79 % of variance). For



123

and the highest in Og-BRI (0.283) (Table 3). O. grandi-

flora showed a higher UST value (0.119) than O. crassifo-

lia. The values of genetic variability within the populations

were high (88 %) and divergence between the populations

was moderate (12 %).

The dendrogram obtained from Nei’s genetic distances

did not indicate structuring into subgroups among the four

populations of O. grandiflora, with the Og-MAC population

being more divergent than the others (Fig. 2b). The PCO,

however, demonstrated structuring in a circular gradient,

where Og-MAC and Og-AMB appeared as the most

genetically distinct populations in relation to each other,

with Oc-CAR totally overlapping Og-BRI and Og-AMB

(Fig. 3b). Approximately 44 % of the species variation was

explained in the first two axes of the PCO. The LnP(K) and

DK values obtained by Bayesian structure analysis indi-

cated the presence of two gene pools, one composed prin-

cipally of the Og-MAC population and distinct from the

others, especially from Og-AMB (Fig. 4b).

Sympatric populations in the Serra do Caraca

First two axes of the PCO explained 52.26 % of the varia-

tion, with the two populations being strongly separated on

axis 1 (Fig. 3c). Octomeria crassifolia demonstrated greater

dispersion than O. grandiflora, apparently reflecting the

higher genetic variability present in that population

(Table 3). AMOVA indicated that 18 % of the genetic

variability was due to differentiation between the species,

while 82 % was due to intrapopulational variation. The

LnP(K) and DK values derived from Bayesian structure

analysis indicated the existence of two genetic groups with

clear differentiation between the populations, and without

any evidence of hybridization between the species (Fig. 4c).

Discussion

Variability and genetic structure

The estimates of genetic variability parameters (P, HE, and

I) encountered for the populations of Octomeria crassifolia

and O. grandiflora were moderate to elevated as compared

to many species of orchids (e.g., Sun and Wong 2001; Smith

et al. 2002; Wallace 2002; Silva 2008; George et al. 2009;

Cruz et al. 2011). In a study using co-dominant markers

(allozymes), however, Borba et al. (2001b) reported very

high levels of heterozygosity in species of Acianthera

Fig. 4 Graphic representation

of the different genetic pools

based on ISSR loci obtained by

Bayesian structure analysis of

individuals from a four

populations of Octomeria

crassifolia (65 individuals), for

K = 3; b four populations of O.

grandiflora (54 individuals), for

K = 2; c two sympatric

populations of both species (36

individuals), for K = 2.

Populations are separated by

vertical bars. For population

names see Table 1


123

(Pleurothallidinae), which, like O. crassifolia and O.

grandiflora, are self-compatible and myophilous (princi-

pally if one considers the small variation levels character-

istic of allozyme markers). The genetic diversity levels of

various myophilous species of Bulbophyllum were likewise

observed to be larger than those of the species studied here

(Azevedo et al. 2006, 2007; Ribeiro et al. 2008). The pro-

portions of polymorphic loci and expected heterozygosity

encountered in the populations of O. crassifolia and O.

grandiflora were similar to the averages reported for other

plant species with similar characteristics: herbaceous

perennials, with sexual reproduction, animal pollinated, and

wind-dispersed (Nybom and Bartish 2000; Nybom 2004).

A relatively high genetic structuring was expected in

both Octomeria crassifolia and O. grandiflora, due, in

large part, to pollinator behavior (Loveless and Hamrick

1984). Both orchids are pollinated by dipteran species of

the family Sciaridae (Barbosa et al. 2009) that generally

promote pollination among flowers of the same individual

or in nearby plants (Chase 1985; Borba and Semir 1998).

Additionally, it was expected that Octomeria crassifolia

would demonstrate greater genetic structuring than O.

grandiflora, as populations of the former species occur in

distinct phytophysiognomic environments and show high

morphological differentiation as compared to the morpho-

logical and environmental homogeneity associated with the

latter species. Interestingly, our results demonstrated a high

genetic similarity among the conspecific populations of

both O. crassifolia and O. grandiflora. This high genetic

similarity was similar to that frequently seen in allopatric

populations (Nybom and Bartish 2000) and may be asso-

ciated with the absence (or low numbers) of specific

fragments in the populations, and is consistent with the

allogamic reproduction patterns of these species (Nybom

and Bartish 2000).

In spite of the high genetic similarity among the popu-

lations of O. crassifolia, the greater differentiation of the

Oc-PIE population may be related to the habitat of these

plants, as they grow on ferruginous outcrops and probably

experience environmental pressures distinct from epiphytic

populations. Similar results for plant populations occurring

on ferruginous outcrops were reported by Borba et al.

(2001b) for Acianthera teres (Orchidaceae) and by Lous-

ada et al. (2013) for Vellozia compacta (Velloziaceae). The

greater genetic structuring seen in O. grandiflora is due to

the high genetic differentiation of Og-MAC population,

which is also seen in the genetic distance and other anal-

yses. This population also shows distinct morphological

characters, with larger leaves and flowers than the other

populations.

The high similarity among populations within each of the

two species might be explained by maintenance of gene

flow among the conspecific populations. However, dipteran

pollinators exhibit restricted movement patterns that do not

readily contribute to homogenizing populations (Proctor

et al. 1996; Borba and Semir 2001). One factor that can

contribute to efficient gene flow is seed dispersal, especially

in orchids because of their very light, small, and numerous

seeds (dust diaspora; Dressler 1993). Moreover, it is pos-

sible that a shared historical genetic stock with low differ-

entiation between populations is partly responsible for the

observed similarities between the populations, with incipi-

ent differentiation generating the patterns similar to those

observed in Bulbophyllum species (Ribeiro et al. 2008).

Reproductive isolation between sympatric populations

The observed degree of genetic differentiation between the

Octomeria species is due to the occurrence of exclusive

loci in the two populations, as a consequence of the

absence of gene flow between them which is fostered by

their pollinator specificity (Barbosa et al. 2009). Four

species of Bradysia (Sciaridae) are known pollinators of O.

crassifolia, while only one species of Pseudosciara

(Sciaridae) pollinates O. grandiflora. The species of these

two insect genera are quite distinct in terms of their body

sizes, which probably help guarantee reproductive isolation

of these sympatric plants in cases of pollinator-plant

fidelity lapses (Barbosa et al. 2009). In spite of the fact that

generalist systems are considered the rule in angiosperms

(Herrera 1988; Ollerton 1996; Waser et al. 1996), spe-

cialized pollination systems are frequent in the Orchida-

ceae (Tremblay 1992; Johnson and Steiner 2000). Studies

with multiple conspecific populations have demonstrated

that there can be spatial–temporal variations in pollinator

guilds (Herrera 1988; Dilley et al. 2000), but a multi-

populational study undertaken by Borba and Semir (2001)

reported high pollinator specificity in conspecific popula-

tions of five species of Acianthera that were maintained

throughout the distribution ranges of the species. In the

case of the Octomeria species, in spite of the fact that field

observations were undertaken during only a single season

(Barbosa et al. 2009), the data presented here indicates that

the pollinator specificity observed in these sympatric pop-

ulations is high enough to maintain species’ identities—in

spite of the fact that pollination by dipterans (myophily) is

considered one of the most generalist and promiscuous

pollination syndromes (van der Pijl and Dodson 1966;

Proctor et al. 1996).

Acknowledgments We thank Marcos C. de Melo and Pedro P.

G. Taucce for help with population sampling, and Junia M. Lousada

for help in some laboratory tasks. This work was funded by projects

from the Conselho Nacional de Desenvolvimento Cientıfico e Tec-

nologico (CNPq) and the Fundacao de Amparo a Pesquisa do Estado

de Minas Gerais (FAPEMIG), Brazil. ARB received a fellowship

from CNPq. ELB is supported by a productivity grant from CNPq.


123

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Documents

High genetic variability in self-incompatible myophilous Octomeria (Orchidaceae, Pleurothallidinae) species