Hybridization between two oil-secreting orchids in South Africa

ORIGINAL ARTICLE

Hybridization between two oil-secreting orchids in South Africa

Kim E. Steiner Æ Boni C. Cruz

Received: 29 January 2008 / Accepted: 29 October 2008 / Published online: 22 January 2009

� Springer-Verlag 2009

Abstract Natural hybrids among the specialized terres-

trial oil-secreting orchids of South Africa are extremely

rare even where multiple closely related species co-occur.

We found putative hybrids between Pterygodium catholi-

cum Sw. and P. acutifolium Lindl., two closely related

oil-secreting orchids that lack morphological barriers to

inter-breeding. The purpose of this study was to confirm

the parentage of the putative hybrids using molecular data

from one nuclear (ITS) and two plastid (matK-trnK and

trnL/F introns) DNA regions. Phylogenetic analyses of

sequences as well as nucleotide substitution patterns of the

putative hybrids, putative parents, and their closest rela-

tives were consistent with a hypothesis of hybridization.

We suggest that the hybrids were the result of visits to both

orchids by a single species of oil-collecting bee during a

brief period of flowering overlap. These results suggest that

the rarity of hybrids between these orchids is due to factors

other than genetic incompatibility.

Keywords DNA sequences � Hybridization �Orchidaceae � Pollination � Pterygodium catholicum �Pterygodium acutifolium � Rediviva �Specialization for pollination

Introduction

Some of the most specialized pollination systems in the

world occur among the terrestrial orchids of southern

Africa (South Africa, Namibia, Botswana, Swaziland and

Lesotho), where many species are pollinated by a single

insect species (Johnson and Steiner 2003). This extreme

specificity may explain why few natural hybrids (\4.3%)

have been reported in this relatively large orchid flora of

about 466 species (Linder and Kurzweil 1999). Compared

to the orchid flora of Mediterranean Europe (see Willing

and Willing 1977, 1985), natural hybrids in South Africa,

especially in the Mediterranean-like climate of the Cape

Floral Region, are exceedingly rare. Nearly half of the

approximately 20 reported cases have been between spe-

cies of Disa, the largest (131 species) genus in the flora

(Linder and Kurzweil 1999), and the group with one of the

best-documented examples of adaptive radiation for polli-

nation in the angiosperms (Johnson et al. 1998a). The other

reported hybrids are scattered among a variety of genera in

different tribes or subfamilies (e.g., Satyrium, Habenaria,

Schizochilus, Eulophia and Polystachya). Those that have

been better studied are morphologically intermediate

between their parents, and occur in close proximity to

them (Stewart and Manning 1982; Linder 1990; Ellis and

Johnson 1999).

Southern Africa is also home to some very unusual

looking orchid flowers in genera of Coryciinae sensu lato

(Kurzweil et al. 1991) such as Corycium, Pterygodium,

Ceratandra and Disperis. Many of the 64 species of

Coryciinae that occur in southern Africa secrete non-volatile

oil as a floral reward, and are pollinated by only one or two

species of oil-collecting bees (Steiner 1989, unpublished

research; Whitehead and Steiner 2001; Pauw 2006, 2007).

As one might expect for plants with such a specialized

pollination system, natural hybrids are very rare in the

Coryciinae. Only three putative hybrids, each from a dif-

ferent genus, have been reported for this group. These

include Pterygodium (Lewis 1950), Disperis (Stewart et al.

1982) and Ceratandra (Kurzweil and Archer 2003).

K. E. Steiner (&) � B. C. Cruz

Botany Department, California Academy of Sciences,

55 Music Concourse Dr, San Francisco, CA 94118, USA

e-mail: [email protected]

123

Plant Syst Evol (2009) 277:233–243

DOI 10.1007/s00606-008-0119-7

However, none of these putative hybrids have been verified

and their parentage clearly established.

Two of the most common species of Coryciinae in the

south western Cape of South Africa are Pterygodium cath-

olicum Sw. and P. acutifolium Lindl.. Based on morphology,

these two species, together with P. newdigateae Bolus var.

cleistogamum Bolus, a closely related cleistogamous apo-

mict (Duthie 1916), form an unresolved trichotomy within

section Pterygodium (Linder and Kurzweil 1999). Sister to

the trichotomy is P. platypetalum Lindl. and sister to the rest

of the clade is P. hastatum Bolus. A molecular analysis of

the section confirms the close relationship of these five

species and groups them in a clade that is sister to a clade of

P. cruciferum Sond. and P. connivens Schelpe (Steiner,

unpublished research).

Pterygodium catholicum tends to have flowers that are

greenish white to greenish yellow with a lip appendage that

has serrate margins at its apex, whereas P. acutifolium has

bright yellow flowers with a lip appendage having entire

margins at the apex. Although the geographical range of

the two species is similar, flowering phenology and habitat

specificity differ. P. catholicum flowers mainly in the early

spring and occurs most commonly on nutrient rich clayey

soils bearing renosterveld shrub land vegetation or on

nutrient rich azonal shale bands that occur in otherwise low

nutrient sandy soils bearing mountain fynbos vegetation

(Steiner, unpublished research). P. acutifolium flowers in

late spring and occurs mainly on sandy soils with relatively

low nutrients and fynbos shrubland vegetation (Steiner,

unpublished research). Furthermore, P. catholicum occu-

pies dry habitats, whereas P. acutifolium generally occurs

in seasonally wet areas such as seeps, marshes and stream

banks. Because of the latter two differences, it is rare for

the two species to flower at the same time and to occur in

close proximity. Even in the few areas of fynbos vegeta-

tion, where these two orchids co-occur, they usually flower

only in the first or second season after a fire. Since the

interval between such fires is 4–40 years (Kruger 1983), it

is rare to find these plants in flower together, in any one

year. Thus, the single unverified observation of putative

hybrids by Lewis (1950) may be an indication that

hybridization between these two species is an extremely

rare event or that it is an occasional event that is very rarely

observed. Where P. catholicum occurs in fynbos, it tends to

flower later in the season than it does in renosterveld, and

tends to be more dependent on fire for flowering than in

renosterveld. It is only in situations where P. catholicum

flowers late enough in the season to overlap with the

flowering of P. acutifolium that hybridization between the

two species is possible.

There are additional reasons to believe that hybridiza-

tion between these two species should be a rare occurrence.

P. catholicum is reputed to be pollinated by a single species

of oil-collecting bee, Rediviva peringueyi (Pauw 2006,

2007) and P. acutifolium is pollinated by a different oil-

collecting bee, R. gigas (Whitehead and Steiner 2001).

R. peringueyi is only active in early spring from late

August to early October, whereas R. gigas is active from

mid-October to mid-December (Whitehead and Steiner

1993, 2001). Although most populations of P. catholicum

flower in early spring when Pauw (2006) made his obser-

vations, there are some populations that flower in late

spring (Linder and Kurzweil 1999) when R. peringueyi is

no longer active (Whitehead and Steiner 2001). Therefore,

either the pollinator of P. catholicum is active longer than

previously thought or the late-flowering populations are

pollinated by a different bee.

During the course of fieldwork in 1999 in the Mount

Rochelle Nature Reserve, ‘‘late flowering’’ populations

of P. catholicum and normal flowering populations of

P. acutifolium were found growing in fairly close prox-

imity. In one small population of P. acutifolium, three

plants with morphological characteristics intermediate

between P. acutifolium and P. catholicum were identified.

Only one of the intermediate plants still had old, but intact

flowers. Flowers on the other two plants were partially

eaten and senesced. As judged from the condition of the

flowers on the putative hybrids, these plants had over-

lapped in flowering time both with neighboring plants of

P. acutifolium and with nearby plants of P. catholicum.

Because there were so few putative hybrid plants flower-

ing, it was not possible to gather an adequate sample of

their flowers for a quantitative morphological assessment.

Plants were growing in a small, undisturbed, seasonally

moist, seepage area that is the typical habitat of P. acu-

tifolium in the first spring following a late summer fire. A

small population (\50 plants) of P. catholicum was found

flowering in a much drier site within 25 m of P. acutifo-

lium and the hybrids, and a much larger population of

P. catholicum (c. 200 plants) was found within 450 m. The

purpose of this study was to determine whether molecular

markers could be found that would distinguish the two

parental species and confirm the hybrid identity of the

morphologically intermediate plants.

Study site

The Mount Rochelle Nature Reserve is a large nature

reserve with undisturbed mountain fynbos vegetation in the

steep mountains above the town of Franschhoek in the

Western Cape Province of South Africa. It is contiguous

with the larger Limietberg Nature Reserve (117,000 ha),

and together, they constitute much of the greater Boland

mountain range, an important water catchment area for the

Western Cape.

234 K. E. Steiner, B. C. Cruz

123

Methods

To confirm the hybrid identity of the morphologically

intermediate plants at the Reserve, we sequenced three

DNA regions, two plastid (matK-trnK and trnL/F), and one

nuclear ribosomal internal transcribed spacer (ITS), from

individuals of the two putative parental species, P. cath-

olicum and P. acutifolium, and the three putative hybrid

individuals. We also sequenced the three other closely

related taxa, P. newdigateae var. cleistogamum, P. pla-

typetalum and P. hastatum that occur within a well

supported ‘‘P. catholicum’’ clade together with P. cru-

ciferum and P. connivens (Steiner, unpublished research).

We sampled ten individuals of P. catholicum, five from

Site A consisting of[200 plants, 450 m SW of the Hybrid

Site and five from a population of about 30 plants at Site B

located 920 m N of the Hybrid Site. Ten individuals of

P. acutifolium were sampled from the Hybrid Site. The five

plants of P. catholicum from Site A as well as the three

putative hybrids from the Hybrid Site were collected in

November 1999. The P. acutifolium plants at the Hybrid

Site, as well as the five P. catholicum plants collected at

Site B, were collected in September 2007. Vouchers of the

Pterygodium species as well as one of the hybrids are

deposited in NBG and CAS.

Total genomic DNA was isolated from silica-gel dried

leaf tissue with a DNeasy Plant Mini Kit (Qiagen, Inc.,

Valencia, CA, USA). Prior to extraction, leaf tissue was

ground by high-speed action of the MAX capsule mixer

(Wykle Research, Inc., Carson City, NV, USA). PCR

amplification was performed with standard methods

(Dieffenbach and Dveksler 1995) using BIOLASE (Bioline

USA, Inc., Randolph, MA, USA) or HotStart-IT (USB

Corp., Cleveland, OH, USA) as the DNA polymerase. PCR

products were purified with the ExoSAP-IT PCR Clean-up

system (USB Corp.). Cycle sequencing was performed

with the ABI Prism BigDye Terminators v3.1 Cycle

Sequencing Reaction kit (Applied Biosystems, Foster City,

CA, USA) by using 1/8-scale reaction mixtures in a

model 9600 PCR System thermal cycler (Perkin-Elmer,

Boston, MA, USA) or Bio-Rad MyCycler thermal cycler

system (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Sequences were generated with an ABI Prism 3100 genetic

analyzer or an ABI Prism 3130xl genetic analyzer (Applied

Biosystems) by obtaining forward and reverse reads for all

samples. Forward and reverse sequences were edited using

the computer program Sequencher 4.1.2 (Gene Codes

Corp., Ann Arbor, MI, USA). Sequences were aligned

manually and gaps introduced into the alignment were

treated as missing data. Polymorphisms among the ITS

sequences were recognized as clear overlapping secondary

peaks in both forward and reverse strands. Following

Fuertes Aguilar et al. (1999), we distinguished two types of

polymorphism: normal polymorphisms, where two peaks

overlapped, and the signal for the secondary peak was at

least 70% as strong as that of the predominant peak and

asymmetric polymorphisms where the secondary peak was

\70% as strong as the predominant peak. Asymmetric

polymorphisms were scored for the base with the strongest

signal, whereas normal polymorphisms were scored using

the IUPAC ambiguity symbols. The aligned sequences

were analyzed using maximum parsimony in PAUP 4.0b10

(Swofford 1998). The shortest MP tree was found using a

heuristic search (100 replicates with random sequence

addition to generate the starting tree in each replicate).

TBR branch swapping was used with the ‘‘steepest

descent’’ option off. Clade support was obtained with

nonparametric bootstrap analysis in PAUP (1,000 pseu-

doreplicates with 10 random sequence addition replicates

in each and maxtrees set at 5,000). TBA branch swapping

was used with the ‘steepest descent’ option off and the

‘multrees’ option on.

Amplification and sequencing of the ITS region

employed primers ITS-4 (50-TAT GCT TAA ACT CAG

CGG GT-30), ITS-5p (50-GGA AGG AGA AGT CGT AAC

AAG G-30), ITS-2p (50-GCT GCG TTC TTC ATC GAT

GC-30), and ITS-3p (50-GCA TCG ATG AAG AAC GCA

GC-30) from Swensen et al. (1998). The matK-trnK region

was amplified with the primers matK-19F (50-CGT-TCT

GAC CAT ATT GCA CTA TG-30) from Pridgeon et al.

(2001) and trnK2R (50-AAC TAG TCG GAT GGA GTA

G-30) from Johnson and Soltis (1995). Sequencing of

matK-trnK was performed with the two amplification

primers and the internal primers matK832*R (50-ACA

TAA TGT ATG AAA GGW TCT TTG A-30) (Whitten

et al. 2000) and matK731F (50-TCT GGA GTC TTT CTT

GAG CGA-30) (Pridgeon et al. 2001). The trnL-trnL/F

region was amplified and sequenced with the universal

primers c (50-CGA AAT CGG TAG ACG CTA CG-30), d

(50-GGG GAT AGA GGG ACT TGA AC-30), e (50-GGT

TCA AGT CCC TCT ATC CC-30), and f (50-ATT TGA

ACT GGT GAC ACG AG-30) (Taberlet et al. 1991). Target

sequences unsuccessfully amplified with the external

primers were occasionally amplified in two fragments with

one external and one internal primer. Sequences used in

this study have been deposited in GenBank (see Table 1 for

accession numbers). The alignment of the sequences is

available from the corresponding author.

Results

Plants of a putative hybrid and its two parental species

from the study area are shown in Fig. 1a–c. The most

obvious differences between the flowers of the two parents

are the shape and color of the corolla. P. catholicum at the

Hybridization between oil-secreting orchids 235

123

study site has greenish yellow flowers that are elliptical in

outline with the edges of the lateral petals flared outward,

whereas P. acutifolium at the study site has bright yellow

flowers that are broadly ovate with the lateral petal edges

curving slightly inward. The hybrid flowers are interme-

diate in shape, but with the flaring outward of the lateral

petal edges more similar to P. catholicum, and the lateral

petal color more similar to P. acutifolium (Fig. 1a–c). An

additional morphological character that has not been pre-

viously discussed for P. catholicum and P. acutifolium is

the length of the anther lobe. In P. catholicum, the posterior

(when seen from the side) anther lobe is much shorter than

the anterior rostellum lobe that bears the viscidium,

whereas in P. acutifolium the anther lobe is nearly equal in

height to the rostellum lobe (Fig. 2d, f). In the hybrid, the

size of the anther lobe is intermediate in height (i.e., larger

than in P. catholicum, but smaller than in P. acutifolium

(Fig. 2e).

Insertion/deletions: Both populations of P. catholicum

and the related P. newdigateae had a 14-bp deletion in the

matK-trnK sequence relative to the other three species

examined. There was also a 14-bp deletion among the trnL/

F sequences in P. catholicum and P. newdigateae that was

absent from the other species. Also among trnL/F

sequences was a 15-bp insertion found only in P. acutifo-

lium and the hybrids. There were no indels among

ITS sequences that were unique either to P. acutifolium,

P. catholicum or the putative hybrids.

In addition to indels in the chloroplast DNA regions,

there were nucleotide substitutions that serve to distinguish

P. catholicum from P. acutifolium. In the matK-trnK

region, there were nine sites with species-specific character

states, and each of these had the same substitution pattern

in the putative hybrids as in P. acutifolium (Table 1). For

trnL/F region, there were eight sites with species-specific

character states and each of these also had the same sub-

stitution pattern in the putative hybrids as in P. acutifolium

(Table 2). The situation for the nuclear DNA region was

different from that of the chloroplast DNA regions. In

ITS, there were 15 substitutions (10 transitions and 5

Table 1 Character states for variable positions in matK-trnK in which the nucleotide is species specific for Pterygodium acutifolium and P.catholicum

Pterygodium spp. Coll. no. Aligned positions for matK-trnK Location Genbank

acc. no.344 658 744 753 1098 1294 1422 1494 1732

P. acutifolium 4281.1 C T C C T A T A C Hybrid Site FJ469823






P. acutifolium 4281a1 C T C C T A T A C Hybrid Site FJ469829




P. ‘‘hybrid’’ 3437.1 C T C C T A T A C Hybrid Site FJ469835



P. catholicum 3435.1 T G G A A C C C A Site A FJ469838





P. catholicum 4282.1 T G G A A C C C A Site B FJ469843





P. newdigateae 3250 T G G A A C C C A Plettenberg Bay FJ469848

P. platypetalum 3365A C G G C T C C A C Riebeck West FJ469849

P. hastatum 3492 C G G C T C C A C Garden Castle FJ469850


123

transversions), mostly from ITS1, that were species specific

(Table 3). At 12 of the 15 sites, two of the three putative

hybrids (3437.2 and 3437.3) had asymmetric polymor-

phisms with the predominant nucleotide the same as the

nucleotide for P. catholicum and the smaller secondary

peak the same as the nucleotide for P. acutifolium

(Table 3). The secondary peak in all cases was \70%

the size of the predominant peak. At the remaining two

sites of these hybrids, no polymorphism was detected, and

the nucleotide was the same as that found in P. catholicum.

For the third putative hybrid (3437.1), all of the 15 species-

specific sites had asymmetric polymorphisms with the

predominant nucleotide the same as that found in

P. acutifolium.

In the parental species P. catholicum, the average

number of polymorphisms detected among the ITS

Fig. 1 Pterygodium spp.

a P. catholicum.

b P. acutifolium 9 catholicum.

c P. acutifolium. Flower close-

ups. d P. catholicum.e P. acutifolium 9 catholicum.

f P. acutifolium. Arrows point

to the posterior anther lobe.

Scale bars a–c = 10 mm,

d–f = 2.5 mm


123

sequences was 2.0 ± 1.5 per sequence, range 1–5

(N = 10) with 70% of these being asymmetric. For P.

acutifolium, the average number of total polymorphisms

detected was 2.3 ± 0.78 per sequence, range 1–4 (N = 10)

with 91% of these being asymmetric. In two of the hybrids,

all polymorphic sites (N = 12) had asymmetric polymor-

phisms. In the third putative hybrid, all 15 sites were

asymmetrically polymorphic. For the putative hybrids, 11

of the polymorphic sites had no corresponding polymor-

phism in individuals of the parental species.

Two of the putative hybrids (3437.2 and 3437.3) appear

to be F1’s based on their intermediate floral morphology

and the additive nature of their polymorphisms (Fig. 1b, e;

Table 3). The third putative hybrid (3437.1) is most likely

a backcross of an F1 to P. acutifolium or possibly an F2

(Table 3). Like the other two hybrids, it has more than five

times the number of polymorphic sites than non-hybrids

and all polymorphisms are additive with one nucleotide

from each parental species. Unlike the two putative F1

hybrids, however, the predominant nucleotides at the

polymorphic sites in hybrid 3437.1 are the same as the

nucleotides from the corresponding sites in P. acutifolium

rather than P. catholicum.

The cladistic analyses of the hybrids and parental spe-

cies reveal discordance between the plastid and nuclear

DNA phylogenies. For the combined plastid DNA regions

(matK-trnK ? trnL/F), the strict consensus of two trees

shows the three hybrids plus one collection of P. acutifo-

lium (4281a.4) in a weakly-supported clade that groups

with the rest of P. acutifolium (Fig. 2). For the ITS phy-

logeny, however, if one codes asymmetric polymorphic

sites for the predominant nucleotide and normal polymor-

phisms as ambiguous using the IUPAC codes, two of the

putative hybrids (3437.2 and 3437.3) group with good

support with P. catholicum ? P. newdigateae var. cleis-

togamum, whereas the third putative hybrid (3437.1)

groups with P. acutifolium (Fig. 3). If one codes all poly-

morphisms among the ITS sequences as ambiguous using

the IUPAC symbols, the hybrids, the parental species and

P. newdigateae var. cleistogamum form a well supported,

but unresolved clade in the strict consensus tree (not

shown). In the combined data of plastid and nuclear DNA

regions, if the asymmetric polymorphic sites of ITS are

coded for the predominant nucleotide and normal poly-

morphic sites are coded as ambiguous, the two putative F1

hybrids (3437.2 and 3437.3) form a clade that is sister to

P. catholicum ? P. newdigateae var. cleistogamum and

the putative backcross or F2 hybrid (3437.1) groups with

P. acutifolium (Fig. 4). However, when all polymorphic

sites of ITS sequences are coded as ambiguous, the com-

bined plastid and nuclear DNA data result in a strict

consensus tree in which the three hybrids group together

with P. acutifolium in a strongly supported, but unresolved,

clade (not shown).

Discussion

This is the first study using molecular markers to verify the

parentage of natural hybrids in the orchid flora of South

Africa. It is significant due to the extremely specialized

nature of orchid pollination systems there, and because

reproductive isolation in these two species is dependent on

phenological and ecological isolation, rather than floral

divergence. The molecular data strongly support the initial

impression from morphological data that the intermediate

plants are hybrids.

The hybrid origin of individuals, populations or species

can be inferred from conflicts between nuclear (i.e., ITS)

and plastid phylogenies or from sequence polymorphisms

in their nrDNA ITS regions (Sang et al. 1995; Fuertes

Aguilar et al. 1999; Gravendeel et al. 2004). Both situa-

tions are apparent in our data. The position of the hybrids

in a partial phylogeny of Pterygodium based on cpDNA

sequences is different from that based on ITS sequences.

Furthermore, the presence of the same chloroplast

Strict consensus tree for mat K-trnK + trnL/F

P. “hybrid” 3437.3

P. “hybrid” 3437.2

P. acutifolium 4281a.4

P. acutifolium 4281.1









P. catholicum 3435.1










P. newdigateae 3250

P. platypetalum 3365A

P. hastatum 3492

63

100

100

100

P. “hybrid” 3437.1

Fig. 2 Pterygodium catholicum clade. Strict consensus of two trees

based on combined plastid DNA sequences (matK-trnK and trnL/F).

Numbers above the branches are bootstrap support values


123

haplotypes in the hybrids as found in P. acutifolium sug-

gests maternal inheritance of the chloroplast genome in

Pterygodium. This is consistent with a report of maternal

inheritance of the chloroplast genome for another orchid in

the subtribe Orchidinae (Caffaso et al. 2005). The three

indels in the chloroplast regions shared by P. acutifolium

and the hybrids and the two substitutions shared by P.

catholicum and the hybrids in the nuclear ITS region,

suggest that the female parent was P. acutifolium and the

male parent was P. catholicum. This is what one would

expect, since the hybrids were found mixed with plants of

P. acutifolium in the seasonally wet habitat that P. acu-

tifolium normally occupies. Presence of the hybrids was not

dependent on an open intermediate or anthropogenically

disturbed habitat. Instead, they were found in an intact area

of the natural mountain fynbos vegetation that was dis-

turbed only by fire, an integral and natural part of the

fynbos ecosystem.

The ITS region is bi-parentally inherited, and hybrids

generally exhibit additivity at nucleotide positions in

which the two parental types differ unless the hybrids

have not been of recent origin (Sang et al. 1995; Fuertes

Aguilar et al. 1999). In the putative hybrids examined in

this study, all of the nucleotide sites that distinguish

P. acutifolium from P. catholicum are polymorphic in one

of the hybrids and all but three of the sites are polymor-

phic in the other two hybrids. These polymorphic sites

have one nucleotide from each parent as one would expect

for a hybrid inheriting copies from both parents. It is not

clear, however, why the predominant nucleotide at the

polymorphic sites of the two putative F1 hybrids, as

indicated by the major peaks in the electropherogram,

were always derived from the paternal parent, P. cathol-

icum. This could be a simple dosage effect caused by a

greater number of ITS copies in P. catholicum than in

P. acutifolium as a result of polyploidy or differences in

the number and location of ribosomal loci. No definitive

chromosome counts have been published for either spe-

cies, but a preliminary count of 2n = c.42 was made for

P. catholicum (Steiner unpublished research). This is also

the base number for the related genus Satryium (Hall

1982).

Table 2 Character states for trnL/F positions in which the nucleotide is species specific for Pterygodium acutifolium and P. catholicum

Pterygodium spp. Coll. no. Positions of aligned trnL/F Location Genbank

acc. no.31 244 256 417 485 780 881 960

P. acutifolium 4281.1 T G C G C A C C Hybrid Site FJ469851






P. acutifolium 4281a1 T G C G C A C C Hybrid Site FJ469857




P. ‘‘hybrid’’ 3437.1 T G C G C A C C Hybrid Site FJ469863



P. catholicum 3435.1 C A A A A C T T Site A FJ469866





P. catholicum 4282.1 C A A A A C T T Site B FJ469871





P. newdigateae 3250 C A A A A C T T Plettenberg Bay FJ469876

P. platypetalum 3365A T A C G C A C C Riebeck West FJ469877

P. hastatum 3492 T A C G C A C C Garden Castle FJ469878


123

For hybrid species, one could account for dominance of

the P. catholicum ribotype through homogenization via

concerted evolution, but the hybrids reported here are

probably F1’s or recent backcrosses and are unlikely to

have had sufficient time for homogenization (but cf.

Fuertes Aguilar et al. 1999). The failure to detect additivity

at two of the parsimony informative sites in two of the

hybrids may simply have been due to the limitation of

direct sequencing to detect rare ITS repeats (Rauscher et al.

2002; Nieto Feliner et al. 2004).

The ability of these two species to hybridize is not

surprising, since breeding barriers among Orchidaceae are

often weak, and the morphological similarity of these two

species suggests a very close relationship (but cf. Johnson

et al. 1998b) (Fig. 1a–f). Both species secrete oil from the

apex of the lip appendage, have pollinaria on each side of

the lip appendage at the base, and have the stigma hidden

behind the base of the lip appendage. Furthermore, the

position of the viscidium in the outer rostellum lobe and its

resultant point of attachment to the bee is the same for each

bee species, despite minor differences in shape and the

presence or absence of a conspicuous anther lobe adjacent

to the lobe of the rostellum. Likewise, the viscidium of the

pollinarium adheres to the inner distal portion of the bas-

itarsus of the middle legs in both bee species (Pauw 2006,

Steiner unpublished research). The reciprocal cross with P.

catholicum as the female parent and P. acutifolium as the

pollen plant may also be possible as a few preliminary

crosses produced viable-looking seed in field-collected

plants (Steiner, unpublished research). This cross would be

less likely in nature, since P. catholicum generally flowers

before P. acutifolium.

It is surprising to find P. catholicum and P. acutifolium

occurring in close proximity and flowering with sufficient

Table 3 Pterygodium acutifolium and P. catholicum character states for ITS1, 5.8S, and ITS2 positions in which the state is species specific

Pterygodium spp. Coll. no. Aligned positions for ITS Location Genbank

acc. no.ITS1 5.8s ITS2

12 13 43 53 111 115 172 178 201 207 244 390 614 617 618

P. acutifolium 4281.1 T C A A A T C/t C A G T T G C T Hybrid Site FJ469879






P. acutifolium 4281a1 T C A A A T C/t C A G T T G C T Hybrid Site FJ469885




P. ‘‘hybrid’’ 3437.1 T/c C/a A/t A/g A/c T/c C/t C/t A/g G/a T/c T/c G/t C/g T/c Hybrid Site FJ469891

P. ‘‘hybrid’’ 3437.2 C/t A/c T/a G C/a C/t T T/c G/a A/g C/t C/t T/g G/c C/t Hybrid Site FJ469892

P. ‘‘hybrid’’ 3437.3 C/t A/c T/a G C/a C/t T T/c G/a A/g C/t C/t T/g G/c C/t Hybrid Site FJ469893

P. catholicum 3435.1 C A/t T G C C T T G A C C T G C Site A FJ469894

P. catholicum 3435.2 C A T G C C T T G A C C T G C Site A FJ469895




P. catholicum 4282.1 C A T G C C T T G A C C T G C Site B FJ469899





P. newdigateae 3250 C A T G C C T T G A C C T G C Plettenberg Bay FJ469904

P. platypetalum 3365A T C T G G T T C A G T C C G C Riebeck West FJ469905

P. hastatum 3492 T C T G G T C T A C T C G C T Garden Castle FJ469906

Positions with two nucleotides listed are asymmetrically polymorphic with the predominant nucleotide (nucleotide with strongest signal)

capitalized and appearing first


123

overlap for a pollinator to transfer pollen between them.

The two parental species rarely co-occur, and have modally

different flowering times. This may explain why there has

been little obvious selection for divergence in floral mor-

phology and/or sterility barriers between them. Our results,

however, lend credence to the report of hybridization

between these two species by Lewis (1950), and we suspect

that hybrids might also be found in at least one or two

additional localities where the senior author has seen the

two species growing in close proximity (Steiner, unpub-

lished research). The identification of additional sites

where these two species hybridize, however, is hampered

by the long intervals between flowering of these fire-

dependent species. In other populations where two or more

oil-secreting orchids commonly co-occur, there appears to

have been selection to minimize reproductive interference

through divergence in the relative positioning of pollinaria

and stigmas within the flower. This results in differential

placement of pollinaria on the bee. For example, in

Disperis (Steiner 1989) and some Pterygodium species

(Pauw 2006), co-occurring species share the same pollin-

ators, but place pollinaria on different parts of the bee’s

body (e.g., forelegs, midlegs or tip of the abdomen) and,

thus, reduce the chance of hybridization.

The pollination event that produced the hybrids must

have taken place at least 9 years prior to this study, since

these plants are dependent on fire for flowering and gen-

erally flower only 1–2 years after fire. The previous

burning of this site occurred in 1989 (Steiner, unpublished

research). The normal interval between fires in fynbos

vegetation is 4–40 years (Kruger 1983). We have no data

on flowering phenology and pollinator abundance at the

hybridization site in 1989, but we did observe the two

species flowering in close proximity nearby (c. 2.5 km

from the hybridization site), and they had a similar pattern

of flowering (P. catholicum flowering before P. acutifolium

with an overlap of 1–2 weeks). We also commonly

observed R. gigas pollinating the flowers of P. acutifolium

at this other site, but only after P. catholicum was mostly

finished flowering. In this case, however, flowers of

Single most parsimonious tree for ITS

P. “hybrid” 3437.2

P. “hybrid” 3437.3


P. catholicum.3435.2









P. newdigateae 3250











P. “hybrid” 3437.1

P. platypetalum 3365A

P. hastatum 3492

98

100

100

Fig. 3 Pterygodium catholicum clade. Single most parsimonious tree

based on ITS sequences. Asymmetric polymorphic characters in the

ITS sequences were coded for the predominant nucleotide and normal

polymorphisms were coded as ambiguous using IUPAC codes.










P. catholicum 4282.4 P. catholicum 4282.5 P. newdigateae

P. “hybrid” 3437.3

P. “hybrid” 3437.2

P. acutifolium 4281a.4 P. “hybrid” 3437.1






P. acutifolium 4281.6 P. acutifolium 4281a.1


P. acutifolium 4281a.3 P. platypetalum 3365A

P. hastatum 3492

100

100

71

76

98

54

Strict consensus tree for mat K-trnK + trnL/F + ITS

Fig. 4 Pterygodium catholicum clade. Strict consensus of two trees

based on combined plastid and nuclear DNA data sets (matK-trnK,

trnL/F and ITS). Asymmetric polymorphic characters in the ITS

sequences were coded for the predominant nucleotide and normal

polymorphisms were coded as ambiguous using IUPAC codes.



123

P. catholicum received no visits and set no seed, whereas

P. acutifolium had a fruit set of 77% (Steiner, unpublished

research).

There are a number of reasons to suggest that the pol-

linator responsible for the interspecific cross between

P. acutifolium and P. catholicum was R. gigas. It is the

only known oil-collecting bee that is active during late

spring when these two species flower (Whitehead and

Steiner 2001). The pollinator of earlier flowering popula-

tions of P. catholicum, R. peringueyi, is known to be active

no later than mid-October in the south western Cape

despite over 15 years of intensive sampling of oil-collect-

ing bees (Whitehead and Steiner 2001, unpublished

research). Furthermore, R. gigas has been observed visiting

P. acutifolium within 2.5 km of the Hybrid Site in other

years. Evidence for the presence of an oil-collecting bee in

the vicinity of both parental species and the hybrids comes

from relatively high fruit set observed in nearby popula-

tions of the parental species. A large population of

P. catholicum c. 450 m SW of the Hybrid Site had a pol-

lination success rate of 39.1% (N = 64 flowers on 18

plants) and a P. acutifolium population of c. 50 plants that

was near Site B (c. 900 m north of Hybrid Site) had a

pollination success rate of 80% (N = 55 flowers on 10

plants) in 1998 (Steiner, unpublished research). Past expe-

rience has shown that only where oil bees are present in a

community, does one find such high levels of fruit set in oil-

secreting host plants (Pauw 2006, 2007; Steiner unpublished

research). The observed pollination of a flower on one of

the putative F1 hybrids, suggests that the pollinator does

not discriminate against these flowers and that the generation

of backcrosses or F2’s is possible in natural populations.

The intricate mechanism by which Pterygodium flowers

are pollinated was correctly predicted by Vogel (1959)

long before pollinators were observed on the flowers. He

recognized that P. acutifolium must be a ‘‘Beinbestauber’’

(leg-pollinator), i.e., the flowers must transfer the pollinaria

from the legs of their pollinators to the concealed stigma

lobes. Furthermore he noted that the lip appendage had a

very smooth surface and that the legs of the pollinators

must occasionally slip off and down into the gap between

the posterior anther lobe and anterior rostellum lobes. He

also predicted that the process of pulling the legs out would

result in contact with the viscidia and extraction of the

pollinaria. Although he only examined the flowers of

P. acutifolium, their close similarity to P. catholicum

flowers caused him to suggest a similar mechanism for that

species. The identity of the pollinators, details regarding

which legs are involved, the exact position of pollinarium

attachment on each leg, and the nature of the floral reward

(i.e., oil rather than nectar) have only recently been rec-

ognized (Whitehead and Steiner 2001, unpublished

research; Pauw 2006). The intricate mechanism described

above argues against pollination by non-specific insects

such as monkey beetles (Scarabaeidae: Hopliini) that have

been implicated in the hybridization of two Disa species in

South Africa (Steiner et al. 1994).

Despite confirmation of natural hybridization between

P. acutifolium and P. catholicum, hybridization among

Coryciinae, that are pollinated by oil-collecting bees, is still

less frequent than among related orchids such as Satyrium

(Hall 1982) or Disa (Linder 1981a, b, 1990; Stewart and

Manning 1982) that are pollinated by moths, butterflies or

bees that do not collect oil (Ellis and Johnson 1999;

Johnson 1997; Johnson et al. 1998a). The presence of only

one other report (albeit unsubstantiated) of hybridization

within Pterygodium (Lewis 1950), and the rarity of finding

simultaneous flowering populations of both species in close

proximity, argues against the hypothesis that hybridization

between these two species is common, but overlooked. The

apparent lack of strong genetic barriers to hybridization,

similar floral morphology and pollination mechanisms, as

well as probable pollinator sharing, all suggest that there

should be strong selection against overlapping flowering times

in areas where P. catholicum and P. acutifolium co-occur.

Acknowledgments We thank the Municipality of Franschhoek and

Western Cape Nature Conservation for permission to collect in the

Mount Rochelle Nature Reserve and the Compton Herbarium

(SANBI) for logistical support. We also thank P. Fritsch and Doug

Stone for comments on earlier versions of the manuscript.

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Hybridization between two oil-secreting orchids in South Africa

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