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. 2011 Jan 26;11:28. doi: 10.1186/1471-2148-11-28

A dated phylogeny and collection records reveal repeated biome shifts in the African genus Coccinia (Cucurbitaceae)

Norbert Holstein 1,✉, Susanne S Renner 1
PMCID: PMC3041684  PMID: 21269492

Abstract

Background

Conservatism in climatic tolerance may limit geographic range expansion and should enhance the effects of habitat fragmentation on population subdivision. Here we study the effects of historical climate change, and the associated habitat fragmentation, on diversification in the mostly sub-Saharan cucurbit genus Coccinia, which has 27 species in a broad range of biota from semi-arid habitats to mist forests. Species limits were inferred from morphology, and nuclear and plastid DNA sequence data, using multiple individuals for the widespread species. Climatic tolerances were assessed from the occurrences of 1189 geo-referenced collections and WorldClim variables.

Results

Nuclear and plastid gene trees included 35 or 65 accessions, representing up to 25 species. The data revealed four species groups, one in southern Africa, one in Central and West African rain forest, one widespread but absent from Central and West African rain forest, and one that occurs from East Africa to southern Africa. A few individuals are differently placed in the plastid and nuclear (LFY) trees or contain two ITS sequence types, indicating hybridization. A molecular clock suggests that the diversification of Coccinia began about 6.9 Ma ago, with most of the extant species diversity dating to the Pliocene. Ancestral biome reconstruction reveals six switches between semi-arid habitats, woodland, and forest, and members of several species pairs differ significantly in their tolerance of different precipitation regimes.

Conclusions

The most surprising findings of this study are the frequent biome shifts (in a relatively small clade) over just 6 - 7 million years and the limited diversification during and since the Pleistocene. Pleistocene climate oscillations may have been too rapid or too shallow for full reproductive barriers to develop among fragmented populations of Coccinia, which would explain the apparently still ongoing hybridization between certain species. Steeper ecological gradients in East Africa and South Africa appear to have resulted in more advanced allopatric speciation there.

Background

Clades will typically retain their ecological characteristics, at least over moderate periods of evolutionary time [1,2], and where inherited climatic tolerances are narrow, this will limit species' geographic range expansion. As long as the inherited component of ecological preference is strong, species evolving in allopatry should initially have similar habitat requirements, and ecological differences between them should accumulate gradually [3]. These arguments set up expectations about how climate niches and species ranges in groups of related species should correlate with each other. Phylogeographic analyses of several African plant clades have found strong signal of Neogene habitat fragmentation and opportunity for allopatric speciation [4-8], but provided no quantitative data on ecological requirements of the species involved. Davis et al. [9] in their study of 11 species of the Malpighiaceae genus Acridocarpus showed that aridification in Eastern Africa apparently was accompanied by a small radiation, possibly involving niche shifts, but did not have details on species' drought tolerances. For a clade of tropical African Annonaceae, Couvreur et al. [10] inferred divergence events between East and West African rainforest species during the Pliocene and Miocen, but provided no data on niche shifts. A likely reason for the comparative neglect of tropical African plant groups in eco-evolutionary studies is that ranges are poorly known because the underlying occurrence data are too incomplete [11,12]. Related problems are a lack of monographic studies, poorly understood species boundaries, and few species-level phylogenies, the precondition for identifying sister species.

While African plant clades are thus underrepresented in eco-phylogenetic studies, the immense interest in primate evolution in Africa has resulted in a wealth of data on vegetation and climate history [13-15]. During the Middle Miocene, starting from about 16 Ma onwards, the African continent underwent gradual cooling and uplift in the east and south, leading to an expansion of woodlands and savannas, and reducing the ranges of lowland rain forest species [15-17]. By the Upper Miocene, 7 Ma ago, rifting and volcanism blocked precipitation, amplifying the overall aridification in East Africa [18,19]. The early Pliocene brought slightly warmer climates until c. 3.2 Ma [20], when the African tropics began experiencing dramatic climate changes that lasted throughout the Pleistocene and Holocene [21-24]. During the driest and coolest periods of the Pleistocene (2.6 Ma - 12,000 years ago), rain forests may have been restricted to refugia from which they re-expanded during more favorable periods [25-28]. The Quaternary climate oscillations affected all of equatorial Africa [29], with the most recent catastrophic destruction of rain forest occurring 2500 years ago [30].

Here we investigate clade diversification and changes in species' precipitation niches in the African cucurbit genus Coccinia. Coccinia comprises 27 species (all of them dioecious) and is almost confined to sub-Saharan Africa where it diversified into numerous habitat types. The only species that "escaped" from sub-Saharan Africa is C. grandis, which spread to the highlands of the Arabian Peninsula and tropical Asia, and is now an invasive weed on the Pacific Islands and in the Neotropics [31]. Pollination of Coccinia is by bees [[32]; NH, personal observation in Tanzania, August 2009], including honeybees. The numerous habitat types occupied by its 27 species make Coccinia a suiTable system in which to study niche evolution among close relatives. The niche parameters we focus on are annual precipitation and number of arid months, with species' tolerances being inferred from the occurrences of 1189 geo-referenced collections. Likely past changes in species' ecological preferences were inferred from a time-calibrated molecular tree including all but two of the species. We expected that close relatives would have similar climatic niche envelopes (e.g., drought tolerances), although clearly there had to have been at least two shifts since different Coccinia species occur in semi-arid habitats, woodland, and forest, vegetation types with contrasting precipitation regimes.

Results

Phylogenetic Reconstruction and Divergence Time Estimates

The concatenated plastid DNA alignment comprised 4551 nucleotides from 65 accessions representing 25 of the 27 species of Coccinia. Table 1 lists all DNA sources with their geographic origin, species name and author, and GenBank accession numbers. A maximum likelihood tree (Figure 1) obtained from the plastid data (TreeBASE accession 10846) shows four major groups: A quinqueloba group that occurs in southern Africa, a barteri clade that mostly occurs in Central and West African rain forest, an adoensis clade that is widespread, but absent from Central and West African rain forest, and a rehmannii clade that occurs from Ethiopia via East Africa to southern Africa.

Table 1.

Voucher information and GenBank accession numbers

Species No. Voucher Location matK ndhF- rpl32R IS rpl20- rps12 IS trnL intron trnL- trnF IS trnS- trnG IS LFY 2nd intron ITS
C. abyssinica (Lam.) Cogn. 1 E. Westphal & J. M. C. Westphal-Stevels 1552 (WAG) Ethiopia, Oromia Region HQ608224 HQ608311 HQ608429
C. abyssinica (Lam.) Cogn. 2 E. Westphal & J. M. C. Westphal-Stevels 1951 (WAG) Ethiopia, Oromia Region HQ608312 HQ608385 HQ608368 HQ608430
C. adoensis (Hochst. ex A. Rich.) Cogn. 1 L. E. Davidson 3781 (M) South Africa, Gauteng HQ608226 HQ608274 HQ608314 HQ608396 HQ608396 HQ608432 HQ608195
C. adoensis (Hochst. ex A. Rich.) Cogn. 2 R. Story 6283 (M) Namibia, Otjozondjupa HQ608227 HQ608275 HQ608316 HQ608397 HQ608397 HQ608434 HQ608160 HQ608196 - 8
C. adoensis (Hochst. ex A. Rich.) Cogn. 3 J. Pawek 6124 (MO) Malawi, Northern Region HQ608225 HQ608315 HQ608369 HQ608433
C. adoensis (Hochst. ex A. Rich.) Cogn. 4 R. E. Gereau & C. J. Kayombo 3582 (MO) Tanzania, Iringa HQ608231 HQ608273 HQ608313 HQ608431
C. adoensis (Hochst. ex A. Rich.) Cogn. 5 E. A. Robinson 2944 (M) Zambia, Southern Prov. HQ608228 HQ608318 HQ608398 HQ608398 HQ608436 HQ608199 - 201
C. adoensis (Hochst. ex A. Rich.) Cogn. 6 H. Merxmüller 282 (M) South Africa, Gauteng HQ608229 HQ608319 HQ608370 HQ608437
C. adoensis (Hochst. ex A. Rich.) Cogn. 7 M. Sanane 375 (M) Zambia, Northern Prov. HQ608230 HQ608320 HQ608399 HQ608399 HQ608438
C. adoensis (Hochst. ex A. Rich.) Cogn. 8 A. R. Torre 5337 (M) Mozambique, Zambezia HQ608321 HQ608371 HQ608439
C. adoensis (Hochst. ex A. Rich.) Cogn. 9 D. K. Harder & M. G. Bingham 2584 (MO) Zambia, Lusaka Prov. HQ608268 HQ608299 HQ608364 HQ608492 HQ608191 HQ608221
C. aurantiaca C. Jeffrey 1 M. Richards 20987 (BR) Tanzania, Iringa HQ608235 HQ625507 HQ608401 HQ608401 HQ608443
C. aurantiaca C. Jeffrey 2 P. J. Greenway & Kanuri 14811 (M) Tanzania, Iringa HQ608402 HQ608402 HQ608444 HQ608161 HQ608202
C. aurantiaca C. Jeffrey 3 N. Holstein et al. 86 (M) Tanzania, Dodoma HQ608236 HQ608276 HQ608325 HQ608403 HQ608403 HQ608445 HQ608162
C. aurantiaca C. Jeffrey 4 S. A. Robertson 1925 (MO) Kenya, Eastern Prov. HQ608232 HQ608322 HQ608400 HQ608400 HQ608440
C. barteri (Hook. f.) Keay 1 E. Achigan-Dako 07 NIA 899 (GAT) Guinea, Nzérékoré Region HQ608237 HQ608330 HQ608404 HQ608404 HQ608450 HQ608203
C. barteri (Hook. f.) Keay 2 J. J. Wieringa 6387 (WAG) Gabon, Haut-Ogooué HQ608239 HQ608277 HQ608326 HQ608405 HQ608405 HQ608446 HQ608163 HQ608204
C. barteri (Hook. f.) Keay 3 E. Achigan-Dako 06 NIA 294 (GAT) Guinea, Mamou Region HQ608331 HQ608389 HQ608376 HQ608451
C. barteri (Hook. f.) Keay 4 E. Achigan-Dako 07 NIA 809 (GAT) Ghana, Eastern Region HQ608240 HQ608327 HQ608387 HQ608374 HQ608447 HQ608164
C. barteri (Hook. f.) Keay 5 W. J. J. O. de Wilde et al. 3736 (MO) Cameroon, Central Region HQ608241 HQ608328 HQ608388 HQ608375 HQ608448
C. barteri (Hook. f.) Keay 6 M. A. van Bergen 490 (WAG) Gabon, Ogooué-Maritime HQ608242 HQ608278 HQ608329 HQ608406 HQ608406 HQ608449 HQ608165
C. barteri (Hook. f.) Keay 7 F. J. Fernández-Casas 12077 (MO) Equatorial Guinea, Bioco Island HQ608238 HQ608279 HQ608332 HQ608390 HQ608377 HQ608453
C. grandiflora Cogn. 1 H. Schäfer 05/302 (M) Tanzania, Tanga HQ608243 HQ608280 HQ608333 HQ608407 HQ608407 HQ608454 HQ608166 HQ608205
C. grandiflora Cogn. 2 N. Holstein et al. 98 (M) Tanzania, Tanga HQ608244 HQ608281 HQ608334 HQ608408 HQ608408 HQ608455 HQ608167
C. grandis (L.) Voigt 1 W. J. J. O. de Wilde & B. E. E. Duyfjes 22270 (L) Thailand, Bangkok DQ536651 HQ608282 DQ536537 DQ536762 DQ536762 HQ608456 HQ608168 HQ608207
C. grandis (L.) Voigt 2 R. Müller s.n., Aug. 1999 (MSB) Sudan, Sannar Prov. HQ608335 HQ608409 HQ608409 HQ608457 HQ608169
C. grandis (L.) Voigt 3 H. Schäfer 05/258 (M) Tanzania, Pwani HQ608245 HQ608283 HQ608336 HQ608410 HQ608410 HQ608458 HQ608170 HQ608206
C. heterophylla (Hook. f.) Holstein C. C. H. Jongkind 5905 (WAG) Gabon, Estuaire HQ608246 HQ608337 HQ608411 HQ608411 HQ608459 HQ608171
C. hirtella Cogn. 1 N. Holstein 29 (M) J.-L. Gatard, France, wild source unknown HQ608247 HQ608284 HQ608339 HQ608412 HQ608412 HQ608461 HQ608172
C. hirtella Cogn. 2 S. S. Renner & A. Kocyan 2447 (M) J.-L. Gatard, France, wild source unknown HQ608248 HQ608338 HQ608413 HQ608413 HQ608460
C. keayana R. Fernandes 1 F. C. Straub 140 (BR) Liberia HQ608462
C. keayana R. Fernandes 2 C. C. H. Jongkind et al. 6542 (WAG) Liberia, Grand Gedeh HQ608249 HQ608285 HQ608340 HQ608378 HQ608463 HQ608173 HQ608211
C. longicarpa Jongkind C. C. H. Jongkind 3970 (WAG) Ghana, Ashanti Region HQ608250 HQ608286 HQ608341 HQ608414 HQ608414 HQ608464 HQ608174 HQ608212
C. mackenii Naudin ex C. Huber R. G. Strey 3762 (M) South Africa, Mpumalanga HQ608251 HQ608343 HQ608415 HQ608415 HQ608465
C. megarrhiza C. Jeffrey 1 J. J. F. E. de Wilde 6501 (WAG) Ethiopia, Oromia Region HQ608344 HQ608417 HQ608417 HQ608466
C. megarrhiza C. Jeffrey 2 I. Friis et al. 2664 (MO) Ethiopia, Oromia Region HQ608252 HQ608287 HQ608347 HQ608416 HQ608416 HQ608469 HQ608176
C. megarrhiza C. Jeffrey 3 P. C. M. Jansen 3471 (WAG) Ethiopia, Oromia Region HQ608345 HQ608467
C. megarrhiza C. Jeffrey 4 J. J. F. E. de Wilde 4793 (WAG) Ethiopia, Oromia Region HQ608253 HQ608346 HQ608468 HQ608175
C. microphylla Gilg 1 R. B. Drummond & J. H. Hemsley 4087 (B) Kenya, Coast Province HQ608254 HQ608348 HQ608470 HQ608177
C. microphylla Gilg 2 J. J. F. E. de Wilde & M. G. Gilbert 346 (UPS) Ethiopia, Somali Regional State HQ608255 HQ608349 HQ608418 HQ608418 HQ608471 HQ608178 HQ608213
C. mildbraedii Gilg ex Harms 1 M. Reekmans 7399 (BR) Burundi, Muramvya Prov. HQ608256 HQ608350 HQ608472
C. mildbraedii Gilg ex Harms 2 N. Holstein et al. 76 (M) Tanzania, Morogoro HQ608257 HQ608288 HQ608351 HQ608419 HQ608419 HQ608473 HQ608179
C. ogadensis Thulin M. Thulin et al. 11183 (UPS) Ethiopia, Somali Regional State HQ608258 HQ608289 HQ608352 HQ608474 HQ608214 - 6
C. quinqueloba (Thunb.) Cogn. R. D. A. Bayliss 8470 (M) South Africa, Eastern Cape HQ608259 HQ608290 HQ608353 HQ608420 HQ608420 HQ608475 HQ608180
C. racemiflora Kéraudren 1 I. van Nek 536 (WAG) Gabon, Ogooué-Maritime HQ608355 HQ608421 HQ608421 HQ608477 HQ608182 HQ608217
C. racemiflora Kéraudren 2 J. J. Bos 6590 (WAG) Cameroon, South Prov. HQ608260 HQ608354 HQ608391 HQ608379 HQ608476 HQ608181
C. rehmannii Cogn. 1 S. S. Renner & A. Kocyan 2749 (M) southern Africa, no detailed information DQ536652 HQ608292 HQ625508 DQ536799 DQ536799 HQ608479 HQ608184 HQ608218
C. rehmannii Cogn. var. littoralis A. Meeuse 2 L. E. Codd 9620 (M) South Africa, KwaZulu-Natal HQ608261 HQ625509 HQ608422 HQ608422 HQ608480
C. rehmannii Cogn. var. rehmannii 3 G. Woortman 217 (M) Namibia, Otjozondjupa HQ608262 HQ625510 HQ608392 HQ608380 HQ608481 HQ608185
C. rehmannii Cogn."ovifera" 4 B. de Winter & O. A. Leistner 5598 (M) Namibia, Kunene HQ608263 HQ608291 HQ608356 HQ608423 HQ608423 HQ608478 HQ608183
C. samburuensis Holstein R. B. & A. J. Faden 74/948 (WAG) Kenya, Rift Valley Prov. HQ608264 HQ608293 HQ608357 HQ608393 HQ608381 HQ608482 HQ608186
C. schliebenii Harms 1 E. Westphal & J. M. C. Westphal-Stevels 5539 (WAG) Ethiopia, Oromia Region HQ608294 HQ608358 HQ608483
C. schliebenii Harms 2 G. S. Laizer et al. 1449 (MO) Tanzania, Morogoro HQ608265 HQ608359 HQ608382 HQ608484 HQ608187
C. senensis (Klotzsch) Cogn. 1 N. Holstein et al. 66 (M) Tanzania, Morogoro HQ608266 HQ608295 HQ608360 HQ608424 HQ608424 HQ608485 HQ608188
C. senensis (Klotzsch) Cogn. 2 K. Vollesen MRC4316 (WAG) Tanzania, Lindi HQ608267 HQ608296 HQ608362 HQ608425 HQ608425 HQ608487 HQ608189 HQ608219
C. senensis (Klotzsch) Cogn. 3 A. R. Torre et al. 18788 (MO) Mozambique, Tete HQ608361 HQ608486
C. senensis (Klotzsch) Cogn. 4 E. M. C. Groenendijk et al. 1031 (WAG) Mozambique, Nampula HQ625511 HQ608489
C. senensis (Klotzsch) Cogn. 5 J. Lovett 1597 (MO) Tanzania, Iringa HQ608233 HQ608323 HQ608386 HQ608372 HQ608441
C. senensis (Klotzsch) Cogn 6 C. F. Paget-Wilkes 72 (MO) Tanzania, Iringa HQ608234 HQ608324 HQ608373 HQ608442
C. sessilifolia (Sond.) Cogn. S. S. Renner et al. 2763 (M) Plant grown at Mainz Bot. G. (MJG19-54430); wild source unknown AY968446 HQ608297 DQ648163 AY968568 AY968385 HQ608490 HQ608190 HQ608220
C. spec. nov. C. Geerling & J. Bokdam 662 (MO) Ivory Coast, Bouna area HQ608269 HQ608298 HQ608363 HQ608383 HQ608491
C. subsessiliflora Cogn. H. F. in de Witte 8288 (M) DR Congo, Kivu HQ608270 HQ608365 HQ608395 HQ608384 HQ608493
C. trilobata (Cogn.) C. Jeffrey N. Holstein & P. Sebastian 9 (M) J.-L. Gatard, France, coll. in Kenya HQ608271 HQ608300 HQ608366 HQ608426 HQ608426 HQ608494 HQ608222
Diplocyclos palmatus (L.) C. Jeffrey J. Maxwell s.n. 2 Sep. 2002 Thailand, Chiang Mai DQ536671 HQ608301 DQ536625 DQ536769 DQ536769 HQ608495 HQ608192
Diplocyclos schliebenii (Harms) C. Jeffrey H. J. Schlieben 4363 (M) Tanzania, Kilimanjaro HQ608427 HQ608427 HQ608496 HQ608193 HQ608223
Cucumis hirsutus Sond. N. B. Zimba et al. 874 (MO) Zambia DQ536658 DQ536542 DQ536804 DQ536804 HM597074
Cucumis sativus L. Unknown unknown AJ970307 AJ970307 AJ970307 AJ970307 AJ970307 AJ970307
Peponium vogelii (Hook. f.) Engl. S. S. Renner 2710 (M) Tanzania, Tanga HQ608272 HQ608302 HQ608367 HQ608428 HQ608428 HQ608497 HQ608194
Scopellaria marginata (Bl.) W. de Wilde and Duyfjes A. Kocyan AK178 (BKF) Thailand DQ536751 DQ536612 DQ536804 DQ536804
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Figure 1.

Figure 1

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Phylogeny for Coccinia based on plastid DNA sequence data. Phylogenetic relationships in Coccinia based on 4,551 nucleotides of concatenated plastid DNA sequences obtained for 69 accessions and analyzed under maximum likelihood (ML) and the GTR + Γ model. Numbers below branches refer to ML bootstrap support > 75%. The dots at nodes and behind the two brackets refer to uniquely shared indels (Methods). The naming of the clades follows that in the nuclear tree (Figure 2). A single accession of C. sessilifolia in the nuclear tree groups with the C. quinqueloba clade, but the plastid data do not contain sufficient signal to resolve the placement of this species.

The nuclear LFY 2nd intron alignment (TreeBASE accession 10846) comprised 463 characters for 35 accessions, representing 20 species of Coccinia plus three outgroups. A maximum likelihood tree from these data (Figure 2) does not contradict the plastid tree topology except for a few accessions in the C. adoensis and C. barteri clades discussed below, and an accession of C. sessilifolia, which in the nuclear tree groups with the quinqueloba group, but in the plastid tree groups with the adoensis clade.

Figure 2.

Figure 2

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Phylogeny for Coccinia based on nuclear data. Phylogenetic relationships in Coccinia based on 463 nucleotides of the nuclear LFY 2nd intron, obtained for 35 accessions from 23 species analyzed under maximum likelihood (ML) and the GTR + Γ model. Numbers below branches refer to ML bootstrap support > 75%. The dots at nodes and behind the two brackets refer to uniquely shared indels. The tree was rooted on Peponium vogelii (not shown).

A tree from the nuclear ITS alignment is almost unresolved (data not shown), but ITS sequences helped pinpoint suspected hybridization (Figure 3; see the section on Evidence for Hybridization). For example, individuals of C. adoensis from different parts of the species' range have different ITS sequences.

Figure 3.

Figure 3

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Detail of the nuclear ITS alignment for Coccinia. Section of the aligned Coccinia nuclear internal transcribed spacer (ITS) sequences, indicating gene flow between individuals of C. adoensis with different plastid genotypes.

A chronogram from a slightly reduced plastid DNA data set (Figure 4a) shows the inferred absolute ages (with 95% confidence intervals) for nodes with >0.98 posterior probability. The diversification of Coccinia apparently began 6.9 Ma ago (10.2 - 3.9 Ma, 95% highest posterior density [HPD]), with most of the extant species diversity dating to the Pliocene.

Figure 4.

Figure 4

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Chronogram and ancestral character reconstruction in Coccinia. a. Chronogram for 24 Coccinia species (C. mackenii and C. quinqueloba had identical sequences, and the former was therefore removed; Methods) obtained under a Bayesian strict clock model. Clades are labeled as in Figure 1 and 2; blue bars around mean node ages represent the 95% highest posterior density credibility interval, and are given for all nodes with ≥ 0.98 posterior probability. Pale red backgrounds indicates warm (humid) climate, pale blue backgrounds, cold (arid) climates after [15]. b. Ancestral biome reconstruction on the Bayesian tree obtained from the plastid DNA data for Coccinia; Black = forest, green = woodland and semi-humid savannas, and white = semi-arid habitats. Red arrows indicate biome shifts.

Climatic Tolerances and Biome Preferences among Close Relatives

Differences in climatic tolerances for species in well-supported clades were quantified by pair-wise Mann-Whitney U tests, focusing on annual precipitation and number of arid months (Table 2a - c). After each species or unit (in the case of the three genotypes of C. adoensis) had been assigned to one of three habitat categories (semi-arid habitats, woodland, or forest; see Methods), maximum likelihood inference of habitat shifts on the phylogeny and the Mann-Whitney U tests revealed at least six habitat changes (marked by red arrows in Figure 4b), counting only changes in statistically supported sister species or clades. Differentiation of precipitation preferences within habitat category (e.g., in C. quinqueloba versus C. mackenii, Table 2a) was not counted as a biome shift.

Table 2.

Pairwise Mann-Whitney U tests among species of supported clades in the Coccinia phylogeny

a. Pairwise Mann-Whitney U tests among species of the Coccinia rehmannii clade and the C. quinqueloba group
abyssinica megarrhiza microphylla trilobata rehmannii quinqueloba mackenii hirtella sessilifolia
abyssinica - 0.047*
megarrhiza < 0.001** -
microphylla - < 0.001** 0.971
trilobata < 0.001** - < 0.001**
rehmannii 0.128 < 0.001** -
quinqueloba - 0.004* 0.013* < 0.001**
mackenii < 0.001** - 0.206 < 0.001**
hirtella < 0.001** 0.003* - < 0.001**
sessilifolia < 0.001** < 0.001** < 0.001** -
b. Pairwise Mann-Whitney U tests among species of the Coccinia barteri clade
barteri racemiflora subsessiliflora longicarpa keayana heterophylla spec. nov. mildbraedii
barteri - 0.336 0.001* 0.087 0.056 0.001* 0.006* 0.335
racemiflora 0.009* - 0.026* 0.094 0.091 0.445 0.095 0.601
subsessiliflora 0.63 < 0.001** - 0.027* 0.077 < 0.001** 0.017* 0.045*
longicarpa 0.771 < 0.001** 0.251 - 0.746 < 0.001** 0.011* 0.94
keayana 0.009* 0.968 0.002* 0.012* - < 0.001** 0.026* 0.871
heterophylla 0.016* 0.042* 0.041* 0.018* 0.002* - 0.029* 0.006*
spec. nov. 0.093 0.095 0.017* 0.042* 0.013* 0.941 - 0.003*
mildbraedii 0.004* < 0.001** 0.036* 0.002* < 0.001** 0.148 0.139 -
c. Pairwise Mann-Whitney U tests among species of the Coccinia adoensis clade
senensis aurantiaca adoensis 9 adoensis "pubescens" adoensis grandiflora schliebenii samburuensis ogadensis grandis
senensis - 0.241 0.333 < 0.001** 0.549 < 0.001** < 0.001** 0.333 < 0.001** 0.531
aurantiaca 0.001* - 0.625 < 0.001** 0.1 < 0.001** < 0.001** 0.961 < 0.001** 0.35
adoensis 9 0.444 0.75 - 0.03* 0.281 0.043* 0.071 0.4 0.222 0.705
adoensis "pubescens" < 0.001** 0.352 0.636 - < 0.001** 0.001* 0.15 < 0.001** < 0.001** 0.091
adoensis 0.098 0.01* 0.607 < 0.001** - < 0.001** < 0.001** 0.24 < 0.001** 0.903
grandiflora 0.015* < 0.001** 0.043* < 0.001** < 0.001** - 0.138 < 0.001** < 0.001** < 0.001**
schliebenii < 0.001** < 0.001** 0.071 < 0.001** < 0.001** 0.014* - 0.002* < 0.001** 0.061
samburuensis 0.002* 0.185 0.4 0.061 0.006* < 0.001** < 0.001** - 0.004* 0.521
ogadensis < 0.001** < 0.001** 0.222 < 0.001** < 0.001** < 0.001** < 0.001** 0.004* - 0.002*
grandis 0.02* 0.542 0.914 0.219 0.03* < 0.001** < 0.001** 0.314 < 0.001** -
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Fields above the em dash (-) line are comparisons of the number of arid months, below are comparisons of the annual precipitation. One asterisk (*) indicates significance at the 5% level, two asterisks (**) indicate significance at the 0.1% level. Bold numbers indicate statistically significant differentiation between species (biome switch).

We next tested whether the number of pairs of species that have the same niche preferences differs from that obtained if species habitat distributions were drawn at random (proportion 0.635). Among the 27 nodes in the phylogeny, 6 involved habitat shifts (red arrows in Figure 4), which is significantly fewer than the expected number of 17 (G = 9.4, df = 1, P = 0.0021). Even when the four most basal nodes are deleted from the analysis owing to the ambiguity of their character states, the phylogeny still includes significantly fewer habitat shifts than expected at random (G = 6.5, df = 1, P = 0.011). Thus, occupation of one of our three habitat categories appears to be a statistically conservative trait in the sense that daughter lineages tend to retain habitat type more frequently than expected by chance, given that the random probabilities are estimated from the current distributions of species. The next sections briefly describe the geography and timing of the inferred six shifts between semi-arid habitats, woodland, and forest.

The Coccinia quinqueloba group comprises four species and began diversifying c. 5 Ma (7.5 - 2.4 95% HPD) ago. Its divergence times and habitat preferences are shown in Figure 4, geographic ranges in Figure 5b, and precipitation tolerances in Figure 6. The species in this group occur in two habitat categories (forest and semi-arid habitat), and there was at least one niche shift in terms of the tolerated precipitation regime. The three forest species (of which C. mackenii and C. quinqueloba have identical sequences in 3503 nucleotides) diverged around 2.8 Ma (4.7 - 1.1 95% HPD) ago, during the Late Pliocene to Pleistocene.

Figure 5.

Figure 5

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Distribution of species in the Coccinia barteri clade and the C. quinqueloba clade. a Distribution of species in the Coccinia barteri clade. Dark blue = C. longicarpa, deep blue = C. keayana, pale blue = C. heterophylla, light blue = C. mildbraedii, bright yellow = C. barteri morphs, dark yellow = C. racemiflora, pale yellow = C. subsessiliflora. The arrow marks the Dahomey Gap. b Distribution of species in the quinqueloba group. Dark blue = C. quinqueloba, pale blue = C. mackenii, bright yellow = C. sessilifolia, pale yellow = C. hirtella.

Figure 6.

Figure 6

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Precipitation variation of species in the genus Coccinia. Box-and-whisker plots displaying the median, upper quartile, and lower quartile (box) and range of extremes (whiskers) of the precipitation tolerances (mean annual precipitation and number of arid months) of 1189 georeferenced herbarium collections. Numbers of collections for each species are given in the parentheses following the species names, and the species order follows the phylogeny in Figure 4. Dashed lines separate the clades/groups, and colors indicate the biomes: black = forest, green = woodland and humid open habitats, and white = semi-arid habitats.

The Coccinia barteri clade includes eight Central and West African species plus the East African (Tanzanian) C. mildbraedii (incl. C. ulugurensis); Figure 5a shows the species' geographic ranges (except for Coccinia spec. nov.; Table 1 provides the vouchers and code numbers for each sequenced plant) and Figure 6 their climatic tolerances. Diversification of this clade began 5 Ma (7.6 - 2.7 95% HPD; Figure 4a) ago, that is, at the beginning of the Early Pliocene warming. Most of the species occur in lowland rain forests or mountain forests at elevations up to 2900 m, although C. barteri and C. heterophylla also have been collected in humid semi-deciduous forests and clearings. Coccinia spec. nov. represents a biome shift from rain forest to semi-humid savanna (our woodland category). Coccinia barteri is morphologically diverse, and based on herbarium material, species boundaries in the barteri clade tend to be cryptic (Table 3).

Table 3.

Key characters among forest species in the Coccinia barteri clade, illustrating the high level of morphological differentiation among close relatives (Figure 1 and 2)

Species / accession Male raceme morphology Bracts Calyx teeth Fruit shape Other characters
C. mildbraedii Condensed on long stalk No Upright, short, acute Long cylindrical Tendrils bifid
C. keayana Lax, many-flowered No Erect-reflexed, long, narrow Ovoid Tendrils simple
C. longicarpa Condensed No Erect-upright, broad Long cylindrical Tendrils simple (rarely bifid)
C. heterophylla Condensed (rarely also lax) Yes Upright, long subulate Ovoid Tendrils bifid
C. subsessiliflora Condensed, few-flowered Yes Upright, short, acute Ovoid Tendrils simple; leaves more deeply lobate than in other species
C. racemiflora Lax, many-flowered No Erect-upright, slightly fleshy, short, narrow Ovoid Tendrils bifid
C. barteri (type morph) Condensed, many-flowered Yes Upright, short, acute Ovoid Tendrils simple or bifid
C. barteri 7-like Condensed Yes Reflexed, fleshy, short Ovoid Tendrils simple or bifid
C. barteri 2 Condensed, few-flowered No Erect-reflexed, short, subulate Ovoid Tendrils bifid
C. barteri 4 Condensed, few-flowered Yes Upright, short, acute ? Tendrils simple or bifid
C. barteri 5 Condensed, few-flowered No Erect, short, acute ? Tendrils simple
C. barteri 6 (xracemiflora?) Condensed, but pedicels rather long No Upright, short, acute ? Tendrils bifid
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The Coccinia adoensis clade comprises nine species and includes at least two biome shifts (Figure 4b). The first involves the sister species C. grandiflora and C. schliebenii, which occur in (rain-) forests of East Africa (Figure 4b and 7), while their widespread relative C. adoensis occurs in mountain grasslands, deciduous woodlands, and rarely in moister bushlands (> 450 mm annual precipitation, < 7 months of aridity) from South Africa to Ethiopia and to Nigeria. The deeper split is dated to the Late Pleistocene (c. 2.1 Ma ago), while C. grandiflora/C. schliebenii separated from each other c. 1.3 Ma ago (Figure 4a). The second biome shift involves C. ogadensis and C. grandis, which are adapted to semi-arid conditions (Figure 4b).

The Coccinia rehmannii clade, which started diversifying during an arid period at the end of the Miocene 5.2 Ma (7.9 - 2.8 95% HPD) ago (Figure 4a), comprises five species (Figure 7: blue dots) and two biome switches (Figure 4b). The split between C. abyssinica and C. megarrhiza dates to c. 3.9 Ma ago, at the end of the warm and humid Early Pliocene, and that of C. rehmannii from C. microphylla and C. trilobata to c. 3.2 Ma, during the humid Late Pliocene. The climate tolerances of the five species are shown in Figure 6.

Figure 7.

Figure 7

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Distribution of C. grandiflora, C. schliebenii, and species of the Coccinia rehmannii clade. Bright blue (southern Africa) = C. rehmannii, pale blue = C. trilobata, blackish blue = C. microphylla, ice-blue = C. abyssinica, dark blue = C. megarrhiza. Bright yellow = C. schliebenii, pale yellow = C. grandiflora.

Evidence for Hybridization

One of the incongruities between the nuclear and plastid DNA tree topologies concerns C. racemiflora from rain forests of west equatorial Africa. In the plastid tree (Figure 1), C. racemiflora 1 and C. racemiflora 2 group together and share a 490 bp deletion in trnSGCU-trnGUCC intergenic spacer. Morphologically, these two plants appear to represent the same species. However, in the nuclear LFY tree (Figure 2), C. racemiflora 1 groups with C. barteri 4 while C. racemiflora 2 groups with C. barteri 6. The latter plant is morphologically intermediate between C. barteri and C. racemiflora and may present a hybrid.

Two other incongruities concern C. adoensis (compare accessions 1 to 9 in Figure 1-3). First, in the plastid tree (Figure 1), C. adoensis accessions from East Africa cluster with the East African C. grandiflora and C. schliebenii, while pubescent C. adoensis accessions 1 and 6 from South Africa (originally described as C. pubescens) cluster with a glabrous C. adoensis 9 from southern Zambia. In the nuclear LFY tree (Figure 2), C. adoensis 2, which in the plastid tree clustered with East African plants, instead groups with C. adoensis 9 (South African C. adoensis plants did not yield LFY sequences). The ITS alignment (Figure 3) reveals that single individuals of C. adoensis can have two kinds of sequences: C. adoensis 5 from Zambia (sister to adoensis 2 in the plastid DNA data; Figure 1) harbors sequences matching C. adoensis 1 from South Africa as well as sequences matching C. adoensis 2 from Namibia. Second, in the plastid tree, East African C. adoensis are distant from C. aurantiaca, while in the nuclear tree they are in a polytomy with C. aurantiaca and C. adoensis 9.

Discussion

At the outset of this study, we expected minimally two biome shifts, this being the number required to explain the presence of Coccinia in semi-arid habitats, woodland, and forest. Instead, we found six statistically significant biome shifts among close relatives (marked in Figure 4b). However, this is still fewer than if the habitats were distributed on the phylogeny at random. The onset of Coccinia diversification dates to just 6.9 Ma ago, a time when the warm and humid climate began to become cooler and drier. Climatic conditions then continued to oscillate during the Pliocene and Quaternary (Background). Additionally, the East African rifting led to aridification and more open grasslands starting at 7 - 8 Ma ago [15], [19]. Depending on species' ecological tolerances, these climate fluctuations must have caused range reduction and fragmentation, or expansion and merging. The likely ancestral precipitation preferences of the Coccinia clade remain unresolved (Figure 4b); the sister genus, Diplocyclos, which comprises four species, is restricted to rain forest and semi-deciduous woodlands [31].

The 12 forest species of Coccinia all have discontinuous distributional ranges, as exemplified by C. grandiflora (Figure 7), fitting with forest expansion during Pleistocene interglacials that likely reconnected most forest refugia [33]. Survival in persisting refugia probably explains the populations of C. subsessiliflora in the southern Sudanese Imatong Mts. (Figure 5a), of C. barteri in the mountain region between Zimbabwe and Mozambique, and of C. mildbraedii in the Eastern Arc Mts. For C. heterophylla, which occurs in the Angola Escarpment at 15°30'S (Figure 5a), mist-saturated local vegetation pockets [34] may have offered survival possibilities during dry periods, while the presence of C. mildbraedii in the Kenyan highlands (Figure 5a), may result from introduction by humans (fide label information on the specimen J. B. Gillett 20185, MO, NHT). It nevertheless shows that Central African species would probably find suitable habitats in East Africa if forest expansion advanced further.

The Coccinia barteri clade is interesting in containing two rain forest species (C. longicarpa and C. keayana) with overlapping distributions (Figure 5a) and co-occurrence in the same habitats (e.g., in the Banco Forest Reserve, Abidjan, Ivory Coast). They likely descend from a widespread ancestral species, the range of which became fragmented during the cool/dry mid-Pliocene, with C. longicarpa becoming restricted to southwestern Ghana and C. keayana to Liberia, fitting with Maley's [25] refugia. Today, C. longicarpa is also distributed east of the Dahomey Gap (arrow in Figure 5), an abrupt climatically induced rain forest disjunction in West Africa. Although forest fragmentation during glacial periods likely was severe, present range disruptions in Central and West Africa seem to date only to the recent Holocene [35]. Recurrent fragmentation and reconnection of populations during the Pleistocene apparently led to hybridization and introgression, which would explain the high morphological and genetic variability in C. barteri (Figure 1, Table 3).

Among the few species of Coccinia that appear to have originated during and since the Pleistocene are the forest species C. grandiflora/C. schliebenii from East Africa and C. quinqueloba/C. mackenii (the latter identical in the markers sequenced here) from South Africa. Each pair comprises morphologically similar species with partly overlapping ranges (Figure 5 and 7). The stronger aridity in East and southern Africa compared to Central and West Africa seems to have led to Pleistocene allopatric speciation in these aridity intolerant species. That the range of C. schliebenii extends into Ethiopia and the Didinga Mts. in southeastern Sudan (Figure 7), which have similar amounts of precipitation, probably reflects long-distance seed dispersal by birds [36,37], rather than remnant populations from a once continuous range. This is because intervening forests, such as those of the Usambara Mts. and Mt. Kenya, have been well collected, yet have not yielded C. schliebenii.

Coccinia species of the rehmannii clade and other dry-adapted species occur on either side of the Miombo belt (with 3 - 6 months of aridity), but are absent from the belt itself (Figure 7). The reason does not appear to be the belt's poor lateritic soils [38] since Coccinia species can grow on such soils (C. microphylla: R. Wingfield 1351 and 2893, DSM; C. trilobata: R. Polhill &S. Paulo 962, K), and so are C. grandis (E. Westphal &J. M. C. Westphal-Stevels 1385, MO, WAG; J. J. Lavranos &S. Carter 23258, MO) and C. sessilifolia (G. Germishuizen 9384, MO; S. E. Chadwick 280, MO). Fire is an unlikely explanation too, since Coccinia species have tubers and can re-sprout. During the Pleistocene, the Miombo belt apparently was crossable for ostriches and antelopes [39,40], making its barrier role for Coccinia even more difficult to understand.

Conclusions

The at least six biome shifts among the 27 species of Coccinia analyzed here may be an underestimate because of our assignments of species into just three broad biome types, semi-arid habitats, woodland, and forest. A fuller understanding of the physiological traits behind tolerated precipitation regimes in Coccinia would require transplants or common garden experiments [41]. The present results, based on occurrence data and ecological information from herbarium specimen labels, however show that changes in ecological tolerances (especially drought tolerance) have played an important role in the diversification of Coccinia. A strength of this study is that it is based on consistent species concepts and geo-referenced data for well over 1000 collections.

Methods

Species Distribution Analysis and Biome Coding for Ancestral State Reconstruction

The first author surveyed c. 1400 specimens from 25 herbaria (B, BM, BR, COI, DSM, E, EA, FT, GAT, GOET, H, HBG, HEID, K, M, MO, MSB, NHT, P, S, UBT, WAG, and partly C, LISC, and LISU). Collecting localities (and some ecological information) were taken from herbarium specimen labels and geo-referenced 1189 of them with Google Earth, Google Maps (Google Inc., Mountain View, CA, USA), and online maps of the Perry-Castañeda Library Map Collection (http://www.lib.utexas.edu/maps/). Climate data were extracted from the WorldClim database (http://www.worldclim.org; [42]) using DIVA-GIS 7.1.6.2 (http://www.diva-gis.org). The number of arid months was calculated by counting how often the arithmetic mean of the monthly minimum and maximum temperature [°C] is larger than half of the monthly precipitation [mm] [43]. For ancestral state reconstruction, we assigned 24 species to one of three habitat categories: Semi-arid habitats (which includes semi-desert, bushlands, semi-arid savannas), woodland (including habitats such as mountain shrublands, humid grasslands, semi-humid savannas), or forest (including semi-deciduous forest, lowland rain forest, and mountain forest). Assignment of specimens/species to habitats followed information given on herbarium specimen labels and the WorldClim data for the respective location. Coccinia grandis, which occurs in African bushlands and savannas as well as in ruderal sites throughout the humid tropics, was coded as "semi-arid habitats" to reflect its drought tolerance.

Differences in annual precipitation and number of arid months tolerated by members of a species pair or a small clade were tested by pair-wise Mann-Whitney U tests in SPSS 13.0 (SPSS, Chicago, IL, USA). Trait reconstructions were carried out in Mesquite 2.71 [44] under maximum likelihood optimization, using the maximum clade credibility tree (with median heights) from the plastid DNA data obtained from BEAST (below) and Lewis' [45] Markov k-state one parameter model.

Finally, we tested whether the number of pairs of species in which members share the same niche preferences differs from that obtained if species habitat distributions were distributed on the tree at random.

Molecular Phylogenetic Taxon Sampling and Methods

We sampled 25 of the 27 species of Coccinia for several plastid and/or nuclear DNA markers. Only the poorly collected species Coccinia pwaniensis Holstein [46] and C. variifolia A. Meeuse could not be included. Trees were rooted on four outgroups, Cucumis sativus, Cucumis hirsutus, Peponium vogelii, and Scopellaria marginata, based on studies that included species from all African genera of Cucurbitaceae [47,48]. DNA was extracted from 3 - 20 mg of leaf tissue from herbarium specimens or silica-dried plant material, using Macherey-Nagel plant extraction kits (Macherey-Nagel, Düren, Germany). For some samples, the lysis buffer was altered by adding sodium meta bisulfite (S9000, Sigma-Aldrich Chemie GmbH, Munich, Germany) to a 10 mM final concentration [49]. PCR reactions used standard conditions, except for the addition of bovine serum albumine (Fermentas, St. Leon-Rot, Germany). We amplified the plastid intergenic spacers trnSGCU-trnGUCC and the rpl20-rps12 using the primers of Hamilton et al. [50], the ndhF-rpl32 spacer using the primers of Shaw et al. [51], the matK gene and trnK intron using the primers of Yokoyama et al. [52], and the trnLUAA intron and trnLUAA-trnFGAA spacer using the universal primers of Taberlet et al. [53]. PCR products were checked on a 1% agarose gel, and those with multiple bands were run on a 2% agarose gel, cut, and treated with the Wizard SV PCR clean-up kit (Promega GmbH, Mannheim, Germany), following the manufacturer's instructions. Phusion high fidelity DNA Polymerase (Finnzymes, Espoo, Finland) was used for recalcitrant and low-concentrated samples and to amplify the 2nd intron in the nuclear LFY gene. Primers for this region came from Volz and Renner [54] and from a M.Sc. thesis carried out in our lab [55]: LFYubiq F1: 5'-CAY CCN TTY ATH GTN CAN GAR CC-3'; LFYubiq-R1: 5'-GCR TAR CAR TGN ACR TAR TGN CKC AT- 3'.

The complete ITS region was amplified using the primers of Balthazar et al. [56]. Where necessary, we used cloning to assess within-plant sequence divergence, focusing on the polymorphic species. For cloning, we ligated PCR products into plasmids of the Promega pGEM-T Vector system (Promega). Plasmids were transformed in ultra competent E. coli DH5alpha strains [57]. Positive (white) plasmid colonies were picked from the ampicillin blue/white selection agar plates, solved in 4 ml LB medium with 100 mg/ml ampicillin, and grown over-night at 37°C. Plasmids were obtained using GeneElute Miniprep Kit (Sigma-Aldrich) and directly amplified with primer oligonucleotides and settings as mentioned above. PCR products were purified and sequenced, using the same primers. Sequencing was performed on an ABI Prism 3130 Avant capillary sequencer using Pop-7 polymer (Applied Biosystems, Foster City, CA, USA), and sequences were edited with Sequencher v. 4.6 (Gene Codes, Ann Arbor, MI, USA).

Alignment, Phylogenetic Inference and Divergence Time Estimation

Sequences were aligned by eye, using MacClade v. 4.06 [58]. We excluded ambiguously alignable regions and structurally homoplastic sections. This concerned a total of 219 alignment positions in the plastid data (mostly microsatellites) and 42 nucleotides in the nuclear LFY matrix. Tree inference relied on maximum likelihood and was carried out in RAxML v. 7.2.2 [59], with the final parameter evaluation done under the GTR + Γ substitution model. We used this model to approximate the best-fit models found with Modeltest v. 3.7 [60], which under a hierarchical likelihood ratio test indicated the F81 + I + Γ model as the best fit for the combined plastid data, while under the Akaike information criterion it found TVM + I + Γ as the best fit. Statistical support for individual nodes was assessed via bootstrapping with 100 replicates [61]. Nucleotide insertions and deletions (indels) were plotted on the resulting tree to test whether they contained phylogenetic information.

Divergence times were inferred using the program BEAST v. 1.5.2 [62], which employs a Bayesian Markov chain Monte Carlo (MCMC) approach to co-estimate topology, substitution rates, and node ages. The input data consisted of a matrix comprising 26 accessions from 24 species of Coccinia (C. mackenii and C. quinqueloba had identical sequences, and the former was therefore removed) and six outgroup accessions. There are no Coccinia fossils, and we therefore used a secondary calibration from a fossil-calibrated Cucurbitaceae-wide dating analysis [48] that obtained an age of 15 million years (Ma), with a standard deviation of 3.0 Ma, for the split between Coccinia and Diplocyclos. We used this age as a prior constraint on the root node of Coccinia, with a normal distribution and a standard deviation (SD) of 2.6 Ma. A SD of 2.7 Ma (or larger) resulted in poor convergence of the MCMC chain. We used a strict clock model (which we preferred because we have a single secondary constraint), a Yule process tree prior, and MCMC chains of 10 million generation length, with parameters sampled every 1000th generation. The first 20% of the trees was discarded as burn-in, and convergence and mixing of the chain were assessed by consistency across runs, inspection of trace plots in the program TRACER v. 1.4.1 [62], and from the effective sample sizes (ESS), which were well above 1000 for all estimated parameters. Four independent BEAST runs yielded the same maximum clade credibility topology, and we also ran an analysis without the data to verify that the effective priors do not contradict the original priors and to assess the informativeness of the data. The cut-off for nodes to be considered in the chronogram was ≥ 0.98 posterior probability.

Authors' contributions

NH generated sequences and alignments, distribution data, performed data analyses, and worked on the manuscript. SSR conceived the study and drafted the manuscript. Both authors read and approved the final manuscript.

Contributor Information

Norbert Holstein, Email: holstein@lrz.uni-muenchen.de.

Susanne S Renner, Email: renner@lrz.uni-muenchen.de.

Acknowledgements

We thank R. E. Ricklefs for help with statistics and two reviewers whose insightful comments greatly improved our manuscript. The work was supported by the German Research Council (DFG RE603/6-1).

References

  1. Wiens JJ, Graham CH. Niche conservatism: integrating evolution, ecology, and conservation biology. Annual Review of Ecology, Evolution, and Systematics. 2005;36:519–539. doi: 10.1146/annurev.ecolsys.36.102803.095431. [DOI] [Google Scholar]
  2. Crisp MD, Arroyo MTK, Cook LG, Gandolfo MA, Jordan GJ, McGlone MS, Weston PH, Westoby M, Wilf P, Linder HP. Phylogenetic biome conservatism on a global scale. Nature. 2009;458:754–756. doi: 10.1038/nature07764. [DOI] [PubMed] [Google Scholar]
  3. Peterson AT, Soberón J, Sánchez-Cordero V. Conservatism of ecological niches in evolutionary time. Science. 1999;285:1265–1267. doi: 10.1126/science.285.5431.1265. [DOI] [PubMed] [Google Scholar]
  4. Dauby G, Duminil J, Heuertz M, Hardy OJ. Chloroplast DNA polymorphism and phylogeography of a Central African tree species widespread in mature rainforests: Greenwayodendron suaveolens (Annonaceae) Tropical Plant Biology. 2010;3:4–13. doi: 10.1007/s12042-010-9041-6. [DOI] [Google Scholar]
  5. Born C, Alvarez N, McKey D, Ossari S, Wickings EJ, Hossaert-McKey M, Chevallier M-H. Insights into the biogeographical history of the Lower Guinea forest domain: evidence for the role of refugia in the intraspecific differentiation of Aucoumea klaineana. Molecular Ecology. 2011;20:131–142. doi: 10.1111/j.1365-294X.2010.04919.x. [DOI] [PubMed] [Google Scholar]
  6. Lowe AJ, Harris D, Dormontt E, Dawson IK. Testing putative African tropical forest refugia using chloroplast and nuclear DNA phylogeography. Tropical Plant Biology. 2010;3:50–58. doi: 10.1007/s12042-010-9045-2. [DOI] [Google Scholar]
  7. Debout GDG, Doucet JL, Hardy OJ. Population history and gene dispersal inferred from spatial genetic structure of a Central African timber tree, Distemonanthus benthamianus (Caesalpinioideae) Heredity. 2011;106:88–99. doi: 10.1038/hdy.2010.35. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Muchugi A, Lengkeek AG, Kadu CAC, Muluvi GM, Njagi ENM, Dawson IK. Genetic variation in the threatened medicinal tree Prunus africana in Cameroon and Kenya: Implications for current management and evolutionary history. South African Journal of Botany. 2006;72:498–506. doi: 10.1016/j.sajb.2006.01.003. [DOI] [Google Scholar]
  9. Davis CC, Bell CD, Fritsch PW, Mathews S. Phylogeny of Acridocarpus-Brachylophon (Malpighiaceae): implications for Tertiary tropical floras and Afroasian biogeography. Evolution. 2002;56:2395–2405. doi: 10.1111/j.0014-3820.2002.tb00165.x. [DOI] [PubMed] [Google Scholar]
  10. Couvreur T, Chatrou L, Sosef MSM, Richardson J. Molecular phylogenetics reveal multiple tertiary vicariance origins of the African rain forest trees. BMC Biology. 2008;6:54. doi: 10.1186/1741-7007-6-54. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kier G, Mutke J, Dinerstein E, Ricketts TH, Küper W, Kreft H, Barthlott W. Global patterns of plant diversity and floristic knowledge. Journal of Biogeography. 2005;32:1107–1116. doi: 10.1111/j.1365-2699.2005.01272.x. [DOI] [Google Scholar]
  12. Küper W, Sommer JH, Lovett JC, Barthlott W. Deficiency in African plant distribution data - missing pieces of the puzzle. Botanical Journal of the Linnean Society. 2006;150:355–368. [Google Scholar]
  13. Pickford M. Uplift of the roof of Africa and its bearing on the evolution of mankind. Human Evolution. 1990;5:1–20. doi: 10.1007/BF02436472. [DOI] [Google Scholar]
  14. Kingston JD, Hill A, Marino BD. Isotopic evidence for Neogene hominid paleoenvironments in the Kenya Rift Valley. Science. 1994;264:955–959. doi: 10.1126/science.264.5161.955. [DOI] [PubMed] [Google Scholar]
  15. van Zinderen Bakker EM, Mercer JH. Major late Cainozoic climatic events and palaeoenvironmental changes in Africa viewed in a world wide context. Palaeogeography, Palaeoclimatology, Palaeoecology. 1986;56:217–235. doi: 10.1016/0031-0182(86)90095-7. [DOI] [Google Scholar]
  16. Axelrod DI, Raven PH. In: Biogeography and Ecology of Southern Africa. Werger MJA, editor. The Hague: W. Junk bv Publishers; 1978. Late Cretaceous and Tertiary vegetation history of Africa; pp. 77–130. [Google Scholar]
  17. Jacobs BF. Palaeobotanical studies from tropical Africa: relevance to the evolution of forest, woodland and savannah biomes. Philosophical Transactions of the Royal Society London - B. 2004;359:1573–1583. doi: 10.1098/rstb.2004.1533. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Cerling TE, Harris JM, MacFadden BJ, Leakey MG, Quade J, Eisenmann V, Ehleringer JR. Global vegetation change through the Miocene/Pliocene boundary. Nature. 1997;389:153–158. doi: 10.1038/38229. [DOI] [Google Scholar]
  19. Sepulchre P, Ramstein G, Fluteau F, Schuster M, Tiercelin J-J, Brunet M. Tectonic uplift and Eastern Africa aridification. Science. 2006;313:1419–1423. doi: 10.1126/science.1129158. [DOI] [PubMed] [Google Scholar]
  20. Zachos J, Pagani M, Sloan L, Thomas E, Billups K. Trends, rhythms, and aberrations in global climate 65 Ma to present. Science. 2001;292:686–693. doi: 10.1126/science.1059412. [DOI] [PubMed] [Google Scholar]
  21. deMenocal PB. Plio-Pleistocene African climate. Science. 1995;270:53–59. doi: 10.1126/science.270.5233.53. [DOI] [PubMed] [Google Scholar]
  22. Dupont LM, Jahns S, Marret F, Ning S. Vegetation change in equatorial West Africa: time-slices for the last 150 ka. Palaeogeography, Palaeoclimatology, Palaeoecology. 2000;155:95–122. doi: 10.1016/S0031-0182(99)00095-4. [DOI] [Google Scholar]
  23. Gasse F. Hydrological changes in the African tropics since the Last Glacial Maximum. Quaternary Science Reviews. 2000;19:189–211. doi: 10.1016/S0277-3791(99)00061-X. [DOI] [Google Scholar]
  24. Elenga H, Maley J, Vincens A, Farrera I. In: Past climate variability through Europe and Africa. Battarbee RW, Gasse F, Stickley CE, editor. Dordrecht: Kluwer Academic Publishers; 2004. Paleoenvironments, palaeoclimates and landscape development in Atlantic equatorial Africa: a review of key sites covering the last 25 kyrs; pp. 181–198. full_text. [Google Scholar]
  25. Maley J. In: Palaeoecology of Africa and the surrounding islands. Coetzee JA, editor. Vol. 18. Rotterdam, The Netherlands: A. A. Balkema; 1987. Fragmentation de la forêt dense humide africaine et extension des biotopes montagnardes au Quaternaire récent: nouvelles données polliniques et chronologiques. Implications paléoclimatiques et biogéographiques; pp. 307–334. [Google Scholar]
  26. Colyn M, Gautier-Hion A, Verheyen W. A re-appraisal of palaeoenvironmental history in central Africa: evidence for a major fluvial refuge in the Zaire basin. Journal of Biogeography. 1991;18:403–407. doi: 10.2307/2845482. [DOI] [Google Scholar]
  27. Sosef MSM. Begonias and African rain forest refuges: General aspects and recent progress. Dordrecht, The Netherlands: Kluwer Academic Publishers; 1996. [Google Scholar]
  28. Leal ME. Microrefugia, small scale ice age forest remnants. Systematics and Geography of Plants. 2001;71:1073–1077. doi: 10.2307/3668739. [DOI] [Google Scholar]
  29. Marret F, Maley J, Scourse J. Climatic instability in west equatorial Africa during the mid- and late Holocene. Quaternary International. 2006;150:71–81. doi: 10.1016/j.quaint.2006.01.008. [DOI] [Google Scholar]
  30. Maley J. A catastrophic destruction of African forests about 2,500 years ago still exerts a major influence on present vegetation formations. IDS Bulletin. 2002;33:13–30. doi: 10.1111/j.1759-5436.2002.tb00003.x. [DOI] [Google Scholar]
  31. Jeffrey C. In: Flora of Tropical East Africa. Milne-Redhead E, Polhill RM, editor. London: Crown Agents for Oversea Governments and Administrations; 1967. Cucurbitaceae; p. 157. [Google Scholar]
  32. Zimmermann A. Die Cucurbitaceen. Vol. 2. Jena: Gustav Fischer; 1922. [Google Scholar]
  33. Cowling SA, Cox PM, Jones CD, Maslin MA, Peros M, Spall SA. Simulated glacial and interglacial vegetation across Africa: implications for species phylogenies and trans-African migration of plants and animals. Global Change Biology. 2008;14:827–840. doi: 10.1111/j.1365-2486.2007.01524.x. [DOI] [Google Scholar]
  34. Fjeldså J, Lovett JC. Geographical patterns of old and young species in African forest biota: the significance of specific montane areas as evolutionary centres. Biodiversity and Conservation. 1997;6:325–346. [Google Scholar]
  35. Salzmann U, Hoelzmann P. The Dahomey Gap: an abrupt climatically induced rain forest fragmentation in West Africa during the late Holocene. The Holocene. 2005;15:190–199. doi: 10.1191/0959683605hl799rp. [DOI] [Google Scholar]
  36. Bhatt D, Kumar A. Foraging ecology of red-vented bulbul Pycnonotus cafer in Haridwar, India. Forktail. 2001;17:109–110. [Google Scholar]
  37. Bleher B, Potgieter CJ, Johnson DN, Böhning-Gaese K. The importance of figs for frugivores in a South African coastal forest. Journal of Tropical Ecology. 2003;19:375–386. doi: 10.1017/S0266467403003420. [DOI] [Google Scholar]
  38. Walter H, Breckle S-W. Ökologie der Erde. 3. München: Elsevier; 2004. [Google Scholar]
  39. Lorenzen ED, Masembe C, Arctander P, Siegismund HR. A long-standing Pleistocene refugium in southern Africa and a mosaic of refugia in East Africa: insights from mtDNA and the common eland antelope. Journal of Biogeography. 2010;37:571–581. doi: 10.1111/j.1365-2699.2009.02207.x. [DOI] [Google Scholar]
  40. Freitag S, Robinson TJ. Phylogeographic patterns in mitochondrial DNA of the ostrich (Struthio camelus) The Auk. 1993;110:614–622. [Google Scholar]
  41. Araya YN, Silvertown J, Gowing DJ, McConway KJ, Linder HP, Midgley G. A fundamental, eco-hydrological basis for niche segregation in plant communities. New Phytologist. 2011;189:253–258. doi: 10.1111/j.1469-8137.2010.03475.x. [DOI] [PubMed] [Google Scholar]
  42. Hijmans RJ, Cameron SE, Parra JL, Jones PG, Jarvis A. Very high resolution interpolated climate surfaces for global land areas. International Journal of Climatology. 2005;25:1965–1978. doi: 10.1002/joc.1276. [DOI] [Google Scholar]
  43. Gaussen H. VIIIe Congrès international de botanique: 2-14 July 1954. Paris; 1954. Théorie et elassification des climats et microclimats; pp. 125–130. [Google Scholar]
  44. Maddison WP, Maddison DR. Mesquite: a modular system for evolutionary analysis. 2.71 2009. [Google Scholar]
  45. Lewis PO. A likelihood approach to estimating phylogeny from discrete morphological character data. Systematic Biology. 2001;50:913–925. doi: 10.1080/106351501753462876. [DOI] [PubMed] [Google Scholar]
  46. Holstein N, Renner SS. Coccinia (Cucurbitaceae) gains two new species from East Africa, three new synonyms, and one new combination. Kew Bulletin. 2010;65:435–441. doi: 10.1007/s12225-010-9229-9. [DOI] [Google Scholar]
  47. Kocyan A, Zhang L-B, Schaefer H, Renner SS. A multi-locus chloroplast phylogeny for the Cucurbitaceae and its implications for character evolution and classification. Molecular Phylogenetics and Evolution. 2007;44:553–577. doi: 10.1016/j.ympev.2006.12.022. [DOI] [PubMed] [Google Scholar]
  48. Schaefer H, Heibl C, Renner SS. Gourds afloat: a dated phylogeny reveals an Asian origin of the gourd family (Cucurbitaceae) and numerous oversea dispersal events. Proceedings of the Royal Society B: Biological Sciences. 2009;276:843–851. doi: 10.1098/rspb.2008.1447. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Horne EC, Kumpatla SP, Patterson KA, Kupta M, Thompson SA. Improved high-throughput sunflower and cotton genomic DNA extraction and PCR fidelity. Plant Molecular Biology Reporter. 2004;22:83a–83i. doi: 10.1007/BF02773352. [DOI] [Google Scholar]
  50. Hamilton MB. Four primer pairs for the amplification of chloroplast intergenic regions with intraspecific variation. Molecular Ecology. 1999;8:521–523. [PubMed] [Google Scholar]
  51. Shaw J, Lickey EB, Schilling EE, Small RL. Comparison of whole chloroplast genome sequences to choose noncoding regions for phylogenetic studies in angiosperms: the tortoise and the hare III. American Journal of Botany. 2007;94:275–288. doi: 10.3732/ajb.94.3.275. [DOI] [PubMed] [Google Scholar]
  52. Yokoyama J, Suzuki M, Iwatsuki K, Hasebe M. Molecular phylogeny of Coriaria, with special emphasis on the disjunct distribution. Molecular Phylogenetics and Evolution. 2000;14:11–19. doi: 10.1006/mpev.1999.0672. [DOI] [PubMed] [Google Scholar]
  53. Taberlet P, Gielly L, Pautou G, Bouvet J. Universal primers for amplification of three non-coding regions of chloroplast DNA. Plant Molecular Biology. 1991;17:1105–1109. doi: 10.1007/BF00037152. [DOI] [PubMed] [Google Scholar]
  54. Volz SM, Renner SS. Hybridization, polyploidy, and evolutionary transitions between monoecy and dioecy in Bryonia (Cucurbitaceae) American Journal of Botany. 2008;95:1297–1306. doi: 10.3732/ajb.0800187. [DOI] [PubMed] [Google Scholar]
  55. Schuster K. The evolution of the Echinopsis huascha clade (Cactaceae), using molecular-phylogenetic methods. München: Ludwig-Maximilians-Universität; 2009. [Google Scholar]
  56. Balthazar Mv, Endress PK, Qiu Y-L. Phylogenetic relationships in Buxaceae based on nuclear Internal Transcribed Spacers and plastid ndhF sequences. International Journal of Plant Sciences. 2000;161:785–792. doi: 10.1086/314302. [DOI] [Google Scholar]
  57. Inoue H, Nojima H, Okayama H. High efficiency transformation of Escherichia coli with plasmids. Gene. 1990;96:23–28. doi: 10.1016/0378-1119(90)90336-P. [DOI] [PubMed] [Google Scholar]
  58. Maddison WP, Maddison DR. MACCLADE: analysis of phylogeny and character evolution. 2.71. Sunderland, MA: Sinauer Associates; 2003. [DOI] [PubMed] [Google Scholar]
  59. Stamatakis A, Ludwig T, Meier H. RAxML-III: a fast program for maximum likelihood-based inference of large phylogenetic trees. Bioinformatics. 2005;21:456–463. doi: 10.1093/bioinformatics/bti191. [DOI] [PubMed] [Google Scholar]
  60. Posada D, Crandall KA. MODELTEST: testing the model of DNA substitution. Bioinformatics. 1998;14:817–818. doi: 10.1093/bioinformatics/14.9.817. [DOI] [PubMed] [Google Scholar]
  61. Stamatakis A, Hoover P, Rougemont J. A rapid bootstrap algorithm for the RAxML web servers. Systematic Biology. 2008;57:758–771. doi: 10.1080/10635150802429642. [DOI] [PubMed] [Google Scholar]
  62. Drummond A, Rambaut A. BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evolutionary Biology. 2007;7:214. doi: 10.1186/1471-2148-7-214. [DOI] [PMC free article] [PubMed] [Google Scholar]

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