Phylogenetic inference applied to germplasm bank characterization and interspecific breeding in passionfruit

Abstract

Interspecific crosses between the more than 520 Passiflora species may or may not be compatible. The sequences ITS, matK, psbA-trnH, rbcL and trnL-F were used to confirm species identity and to estimate the genetic distances among 48 Passiflora accessions of a germplasm bank. Twenty species were used for crosses within and between distinct Passiflora subgenera. The phylogenetic resolution based on ITS data was superior to that of any single chloroplast marker, recommending it as a DNA barcode for this genus. However, the tree topology based on Bayesian phylogenetic analysis using the combination of the four chloroplast markers possessed greater support for subclades within the subgenus Passiflora, which contains more species of interest for breeders. We could not identify a clear cutoff value of pairwise Kimura two-parameter (K2P) distances to predict success or failure of crosses. Rather, the clustering of species pairs together within the same or closely related phylogenetic subclades predicted the probability of wide cross compatibility more reliably.

Keywords:
Passionfruit; phylogenetics; DNA barcoding; hybrids; identification

INTRODUCTION

Passiflora is the largest genus of the Passifloraceae family, with about 520 described species in tropical and warm temperate regions (MacDougal and Feuillet 2004). Brazil, where about 150 Passiflora species are found, is one of the main centers of diversity of the genus, with 88 endemic species (Cerqueira-Silva et al. 2016, Mezzonato-Pires et al. 2018). Although about 70 species are edible, production chains were only developed for Passiflora edulis Sims. (sour passionfruit) and P. alata Curtis (sweet passionfruit) in Brazil, which is the world’s largest producer and consumer of passionfruit (Cerqueira-Silva et al. 2018).

Wide crossing is considered the most important source of genetic variation for breeding in Passiflora. Disease resistance, ornamental value, self-compatibility, fruit quality and insensitivity to photoperiod are some of the current target traits of Passiflora breeding (Lira Júnior et al. 2014, Ocampo et al. 2016, Cerqueira-Silva et al. 2018). Chances of breeding success depend on the correct identification and classification of species and on basic knowledge about chromosome number and breeding behavior (Hansen et al. 2006). Several biological barriers could play a role in hybrid incompatibility. Usually, genetic distance estimates are used as a first approach to draw conclusions about potential compatibility (Chapman and Burke 2007, Muñoz-Sanz et al. 2020). Knowledge about the intra- and inter-specific genetic variability and phylogenetic relationships between cultivated Passiflora species and their wild relatives may be useful to increase the chances of making wide crosses that produce viable seed and to maintain the molecular polymorphism narrowed by extensive selection (Costa et al. 2012). Several molecular phylogenetic studies have sought to clarify taxonomic issues in Passiflora (Feuillet and MacDougal 2003, Muschner et al. 2003, Hansen et al. 2006, Muschner et al. 2013, Ramaiya et al. 2014, Grisi et al. 2019), which may be the result of the confoundingly high intraspecific variability observed in Passiflora species (Mader et al. 2010).

The Passiflora breeding program of Embrapa Cerrados has exploited hybridizations between wild and cultivated species. Germplasm is maintained at the Active Germplasm Bank Flor da Paixão, in Planaltina-DF, Brazil, one of the largest living collections of Passiflora species. To date, some taxonomic uncertainties still persist, as well as questions on the genetic relatedness and phylogeny of different species of the germplasm included in breeding programs. In this study, DNA sequences of nuclear ribosomal ITS and several chloroplast genes were obtained from a selection of Passiflora species of interest to breeders. The new Passiflora sequences as well as others already available in NCBI GenBank were subjected to phylogenetic analysis to clarify taxonomic questions about these accessions and place them in their phylogenetic context. Thereafter, to help identify potential hybrid combinations for breeding, the correlation between phylogenetic distance and cross-compatibility between Passiflora species was analyzed.

MATERIAL AND METHODS

For the analysis, 48 Passiflora accessions of the Active Germplasm Bank 'Flor da Paixão' of Embrapa Cerrados were selected (Table 1), which are 43 representative species of the current focus of breeding efforts on the improvement of sour, sweet and ornamental passionfruit. To complement the analysis, we used selected reference sequences from recent phylogenetic studies of Passiflora species contained in GenBank, which also served to confirm our previous species identifications through DNA barcodes.

Table 1
Accessions of the genus Passiflora maintained in the ‘Flor da Paixão’ Germplasm Bank at Embrapa Cerrados, selected for phylogenetic analysis and GenBank accession numbers of sequences generated in this study

Nº	Species/variety	Accession No.	ITS	matK	rbcL	psbA-trnH	trnL-F “CF”
P01	P. sidifolia M.Roem.	CPAC-MJ-16-01	KM518260	KM652260	KM652356	KM652308	KM652212
P02	P. amethystina J.C.Mikan “verdadeiro”	CPAC-MJ-13-00	KM518261	KM652261	KM652357	KM652309	KM652213
P03	P. amethystina J.C.Mikan “SP”	CPAC-MJ-13-01	KM518262	KM652262	KM652358	KM652310	KM652214
P04	P. amethystina J.C.Mikan “rui”	CPAC-MJ-13-04	KM518263	KM652263	KM652403	KM652311	KM652215
P05	P. morifolia Mast.	CPAC-MJ-48-01	KM518264	KM652264	KM652359	KM652312	KM652216
P06	P. vitifolia Kunth	CPAC-MJ-46-01	KM518265	KM652265	KM652360	KM652313	KM652217
P07	P. mucronata Lam.	CPAC-MJ-10-06	KM518266	KM652266	KM652361	KM652314	KM652218
P08	P. cerradensis Sacco	CPAC-MJ-45-01	KM518267	KM652267	KM652362	KM652315	KM652219
P09	P. elegans Mast.	CPAC-MJ-44-01	KM518268	KM652268	KM652363	KM652316	KM652220
P10	P. caeruleaL.	CPAC-MJ-14-01	KM518269	KM652269	KM652364	KM652317	KM652221
P11	P. coccinea Aubl.	CPAC-MJ-08-01	KM518270	KM652270	KM652365	KM652318	KM652222
P12	P. actinia Hook.	CPAC-MJ-04-01	KM518271	KM652271	KM652366	KM652319	KM652223
P13	P. foetida L.	CPAC-MJ-28-01	KM518272	KM652272	KM652367	KM652320	KM652224
P14	P. cincinnata Mast.	CPAC-MJ-26-01	KM518273	KM652273	KM652368	KM652321	KM652225
P15	P. odontophylla Harms ex Glaz.	CPAC-MJ-09-01	KM518274	KM652274	KM652369	KM652322	KM652226
P16	P. gardneri Mast.	CPAC-MJ-39-01	KM518275	KM652275	KM652370	KM652323	KM652227
P17	P. bahiensis Klotzsch	CPAC-MJ-58-00	KM518276	KM652276	KM652371	KM652324	KM652228
P18	P. speciosa Gardner	CPAC-MJ-20-01	KM518277	KM652277	KM652372	KM652325	KM652229
P19	P. micropetala Mast.	CPAC-MJ-41-01	KM518278	KM652278	KM652373	KM652326	KM652230
P20	P. malacophylla Mast.	CPAC-MJ-43-01	KM518279	KM652279	KM652374	KM652327	KM652231
P21	P. ambigua Hemsl. ex Hook.f.	CPAC-MJ-49-01	KM518280	KM652280	KM652375	KM652328	KM652232
P22	P. hatschbachii Cervi	CPAC-MJ-50-01	KM518281	KM652281	KM652376	KM652329	KM652233
P23	P. ferruginea Mast.	CPAC-MJ-59-00	KM518282	KM652282	KM652377	KM652330	KM652234
P24	P. coriacea Juss.	CPAC-MJ-60-00	KM518283	KM652283	KM652378	KM652331	KM652235
P25	P. organensis Gardner	CPAC-MJ-51-01	KM518284	KM652284	KM652379	KM652332	KM652236
P26	P. citrina J.M. MacDougal	CPAC-MJ-61-00	KM518285	KM652285	KM652380	KM652333	KM652237
P27	P. sanguinolenta Mast. & Linden	CPAC-MJ-62-00	KM518286	KM652286	KM652381	KM652334	KM652238
P28	P. pentagona Mast.	CPAC-MJ-63-00	KM518287	KM652287	KM652382	KM652335	KM652239
P29	P. tenuifila Killip	CPAC-MJ-30-01	KM518288	KM652288	KM652383	KM652336	KM652240
P30	P. recurva Mast.	CPAC-MJ-64-00	KM518289	KM652289	KM652384	KM652337	KM652241
P31	P. serratodigitata L.	CPAC-MJ-16-02	KM518290	KM652290	KM652385	KM652338	KM652242
P32	P. auriculata Kunth	CPAC-MJ-65-00	KM518291	KM652291	KM652386	KM652339	KM652243
P33	P. edmundoi Sacco	CPAC-MJ-66-00	KM518292	KM652292	KM652387	KM652340	KM652244
P34	P. subrotunda Mast.	CPAC-MJ-17-02	KM518293	KM652293	KM652388	KM652341	KM652245
P35	P. phoenicea Lindl.	CPAC-MJ-53-01	KM518294	KM652294	KM652389	KM652342	KM652246
P36	P. quadriglandulosa Rodschied	CPAC-MJ-67-00	KM518295	KM652295	KM652390	KM652343	KM652247
P37	P. nítida Kunth “MT”	CPAC-MJ-01-07	KM518296	KM652296	KM652391	KM652344	KM652248
P38	P. nítida Kunth “Cerrado”	CPAC-MJ-01-01	KM518297	KM652297	KM652392	KM652345	KM652249
P39	P. suberosa L.	CPAC-MJ-35-01	KM518298	KM652298	KM652393	KM652346	KM652250
P40	P. maliformis L.	CPAC-MJ-68-00	KM518299	KM652299	KM652394	KM652347	KM652251
P41	P. rhamnifolia Mast.	CPAC-MJ-69-00	KM518300	KM652300	KM652395	KM652348	KM652252
P42	P. laurifolia L.	CPAC-MJ-70-00	KM518301	KM652301	KM652396	KM652349	KM652253
P43	P. setacea DC.	CPAC-MJ-12-01	KM518302	KM652302	KM652397	KM652350	KM652254
P44	P. trintae Sacco	CPAC-MJ-40-01	KM518303	KM652303	KM652398	KM652351	KM652255
P45	P. haematostigma Mast.	CPAC-MJ-24-01	KM518304	KM652304	KM652399	KM652352	KM652256
P46	P. edulis Sims “purple”	CPAC-MJ-21-03	KM518305	KM652305	KM652400	KM652353	KM652257
P47	P. edulis Sims “4 stigmas”	CPAC-MJ-M-23	KM518306	KM652306	KM652401	KM652354	KM652258
P48	P. edulis Sims “yellow - Matriz GA2”	CPAC-MJ-M-01	KM518307	KM652307	KM652402	KM652355	KM652259

• * DNA sequence identification refers to the DNA sequence of a Passiflora species used for phylogenetic analysis. This number precedes the species name displayed in the phylogenetic trees.

Genomic DNA was extracted from silica-dried leaf tissue using a CTAB-based protocol (Inglis et al. 2018a) and the nuclear ribosomal internal transcribed spacers (ITS) and four chloroplast regions, trnH-psbA, trnL-trnF, rbcL and matK were sequenced. The procedures and assembly of data matrices were essentially as described previously (Inglis et al. 2018b). A combined matrix of the four chloroplast regions was also prepared, in which alignment gaps indicated unavailable accession data. The ITS of numerous Passiflora accessions was initially unsuitable for Sanger sequencing due to the presence of a strong poly-G-poly-C hairpin loop. However, the quality of the ITS sequence data was high after substituting the dGTP in the original PCR dNTP mix with 7-deaza dGTP (Dierick et al. 1993).

Pairwise Kimura two-parameter (K2P) distances were calculated from a concatenated chloroplast + ITS matrix in MEGA 7 (Kumar et al. 2016), ignoring ambiguous positions. After a tree search in PAUP*, maximum parsimony statistics were generated (v4.0b10; Swofford 2003). Alignment gaps were treated as missing data and heuristic searches comprised 1.000 repeats of five cycles of random taxon addition, holding one tree per cycle, with ACCTRAN character-state optimization and TBR branch swapping. A Bayesian phylogenetic hypothesis based on the ITS matrix and concatenated chloroplast matrix was obtained using reversible-jump MCMC in MrBayes 3.2.2 (Ronquist et al. 2012). Priors were allowed to vary independently for each data partition and one cold and three heated MCMC chains were run in parallel for 10 million generations and sampled every 1000 generations. This runtime was sufficient to cause the convergence diagnostic, the mean standard deviation of split frequencies, to drop to below 0.01 in all repeated runs. The first 25% of the trees were discarded (burn-in period) prior to calculation of 50% majority rule consensus trees. Concordance factors were calculated between the ITS and concatenated chloroplast matrices under the maximum likelihood (ML) criterion in IQTREE (v. 2.1.12; Minh et al. 2020).

For the interspecific hybridizations, 20 Passiflora species, consisting of two cultivated (P. edulis and P. alata) and 18 wild species collected in Brazil, were analyzed. A total of 29 interspecific crosses among the 20 species were attempted as paired combinations (Table 3). Pollination was attempted by direct use of freshly collected pollen on newly opened flowers. The interspecific crosses were classified as compatible or incompatible, according to the resulting fruit and seed set. Compatibility was declared if at least one viable plant resulted from a cross, after seed germination and full plant growth was confirmed in the greenhouse. Incompatibility was declared if an interspecific cross failed to produce viable plants after extensive and repeated pollination attempts under controlled greenhouse conditions.

Table 2
Character and maximum parsimony tree statistics

Locus/Marker	n	AL	PIC	CC	CFI/NCFI	TL	CI	RI	RC
nrITS	95	828	387	324	57/0.620	2027	0.4435	0.7945	0.3524
matK	63	725	126	519	41/0.683	320	0.7906	0.914	0.7226
psbA-trnH	76	690	135	479	42/0.575	460	0.6348	0.8549	0.5427
rbcL	147	551	106	415	54/0.375	320	0.5313	0.8895	0.4726
trnLF	154	1179	191	790	77/0.510	682	0.7273	0.9086	0.6608
Combined Chloroplast	149	3094	540	2185	91/0.623	1829	0.6315	0.859	0.5424

• Statistics were generated following heuristic maximum parsimony searches in PAUP*. Where: n= number of included sequences (including reference sequences obtained from GenBank); AL= aligned matrix length; PIC= Parsimony informative characters; CC= Constant characters; CFI= Component information or consensus fork index; NCFI= Nomalized CFI; TL= Tree length; CI = Consistency index; RI = Retention index; RC = Rescaled consistency index.

Table 3
Interspecific crosses, subgenus membership, subclade designation and compatibility reaction of pairs of species used in the study (maternal listed first). *Subgroups are based on the topology of the combined chloroplast DNA Bayesian tree. K2P distances were calculated in MEGA v. 7

#	Species hybridization	Subgenus hybridization	Subclade*	Reaction	K2P distance
1	P. amethystina x P. edulis	Passiflora x Passiflora	2a x 1a	Incompatible	0.0229
2	P. caerulea x P. amethystina	Passiflora x Passiflora	2a x 2a	Compatible	0.0206
3	P. caerulea x P. edulis	Passiflora x Passiflora	2a x 1a	Incompatible	0.0226
4	P. coccinea x P. actinia	Passiflora x Passiflora	1a x 1b	Compatible	0.0221
5	P. coccinea x P. setacea	Passiflora x Passiflora	1a x 1c	Compatible	0.0163
6	P. edulis x P. actinia	Passiflora x Passiflora	1a x 1b	Incompatible	0.0188
7	P. edulis x P. caerulea	Passiflora x Passiflora	1a x 2a	Compatible	0.0226
8	P. edulis x P. setacea	Passiflora x Passiflora	1a x 1c	Compatible	0.0156
9	P. edulis x P. tenuifila	Passiflora x Passiflora	1a x 2a	Incompatible	0.0281
10	P. galbana x P. actinia	Passiflora x Passiflora	1a x 1b	Compatible	0.0189
11	P. galbana x P. alata	Passiflora x Passiflora	1a x 2a	Compatible	0.0229
12	P. galbana x P. edulis	Passiflora x Passiflora	1a x 1a	Compatible	0.0151
13	P. hematoestigma x P. coccinea	Astrophea x Passiflora	6 x 1a	Incompatible	0.0659
14	P. hematoestigma x P. edulis	Astrophea x Passiflora	6 x 1a	Incompatible	0.0652
15	P. laurifolia x P. nitida;	Passiflora x Passiflora	1a x 1a	Compatible	0.0153
16	P. mansoi x P. caerulea	Astrophea x Passiflora	6 x 2a	Incompatible	0.0285
17	P. mansoi x P. edulis	Astrophea x Passiflora	6 x 1a	Incompatible	0.0277
18	P. mucronata x P. alata	Passiflora x Passiflora	2a x 1a	Compatible	0.0224
19	P. mucronata x P. coccinea	Passiflora x Passiflora	2a x 1a	Compatible	0.0223
20	P. quadrangularis x P. alata	Passiflora x Passiflora	1a x 2a	Compatible	0.0098
21	P. sanguinolenta x P. capsularis	Decaloba x Decaloba	3b x 3b	Compatible	0.0105
22	P. sanguinolenta x P. citrina	Decaloba x Decaloba	3b x 3b	Compatible	0.0216
23	P. serratodigitata x P. alata	Passiflora x Passiflora	1d x 2a	Incompatible	0.0198
24	P. serratodigitata x P. coccinea	Passiflora x Passiflora	1d x 1a	Incompatible	0.0226
25	P. serratodigitata x P. edulis	Passiflora x Passiflora	1d x 1a	Incompatible	0.0219
26	P. setacea x P. alata	Passiflora x Passiflora	1c x 2a	Compatible	0.0153
27	P. setacea x P. amethystina	Passiflora x Passiflora	1c x 2a	Compatible	0.0247
28	P. setacea x P. edulis	Passiflora x Passiflora	1c x 1a	Compatible	0.0156
29	P. sidifolia x P. actinia	Passiflora x Passiflora	1b x 1b	Compatible	0.0062

RESULTS AND DISCUSSION

Sequence data representing the entire amplicons of all five markers were successfully generated for all 48 Passiflora accessions selected from the Germplasm Bank (Table 1). Newly sequenced accessions are indicated by the prefix P** in the trees. In the cases of matK and trnH-psbA, many are new sequences of the represented Passiflora species and in the case of ITS, many are now complete ITS1-5.8S-ITS2 records. The phylogenetic analysis based on ITS sequences (Figure 1) and combined chloroplast DNA sequences (matK, trnH-psbA, trnL-F) (Figure 2) allowed the separation of the Embrapa passionfruit accessions into four subgenera known as Passiflora (L.), Decaloba (DC.) Rchb., Astrophea (DC.) Mast. and Deidamioides (Harms) Killip. These results agree with earlier phylogenetic analyses (Muschner et al. 2003, Krosnick et al. 2013), as well as the acknowledged division of the genus into two groups correlated with flower size. The small-flowered group contains species assigned to the subgenera Astrophea, Decaloba and Deidamioides, and the large-flowered group species of the subgenus Passiflora. Some species with small flowers (Passiflora bahiensis, Passiflora malacophylla and Passiflora laurifolia), not analyzed by Muschner et al. (2003), were also found in the latter group. From a horticultural point of view, Passiflora is the most important subgenus to which a large number of species with great variability in flower morphology, size and color are assigned (Cerqueira-Silva et al. 2018), which is also the reason for the greater representation of this group in this study.

Figure 1
Bayesian phylogenetic tree based on ITS sequences. Accessions preceded by an accompanying code (Pxx) were sequenced in this study. Posterior probabilities > 0.9 are given above branches. The boundaries of subgenus Decaloba follow the treatment of Krosnick et al. (2013). Abbreviations for additional subgenera (in bold) are as follows: Pol. = Polyantha; Tet. = Tetrapathea; Dei. = Deidamioides.

Figure 2
Bayesian phylogenetic tree based on combined chloroplast matK, rbcL and trnL-F sequences. Accessions with accompanying code (Pxx) were sequenced in this study. Posterior probabilities > 0.9 are given above branches. The boundaries of subgenus Decaloba follow the treatment of Krosnick et al. (2013). Abbreviations for additional subgenera (in bold) are as follows: Pol. = Polyantha; Tet. = Tetrapathea; Dei. = Deidamioides.

Aside from Bayesian inference (BI) to infer phylogenetic relationships, the performance of each sequenced locus was evaluated under the maximum parsimony (MP) criterion. Results of the five individual markers and a combination of the four chloroplast markers are given in Table 2. The tree inferred by chloroplast marker matK had the highest Consistency Index (CI), but this locus was represented by the fewest Passiflora species in GenBank. Among the markers of Passiflora species with extensive representation in GenBank, chloroplast marker rbcL had the lowest CI of them all, as well as the lowest phylogenetic resolution, as indicated by the low Normalized Consensus Fork Index (NCFI). Combining the chloroplast data into a single concatenated matrix improved the Consensus Fork Index (CFI) compared to the component matrices, but did not raise the CI. Of all markers used, the CI of ITS was the lowest, while the NCFI was higher than all but matK and the combined chloroplast DNA sequences. However, ITS outperformed all individual chloroplast loci and combined chloroplast data in terms of greater tree length, phylogenetic resolution and better pairwise discriminatory power. Nevertheless, the support for subclades within the resolved subgenera of the combined chloroplast tree was superior, particularly in subgenus Passiflora (Figures 1 and 2). The ITS1 portion of the full ITS region has been proposed as a DNA barcode for Passiflora (Giudicelli et al. 2015), in view of the good discriminatory potential across a large representative species sample and the correct identification rate of 68.64%, which can be further improved by the inclusion of ITS2. However, the significant levels of intraspecific variation reported raise concerns with regard to the sole use of ITS sequences for Passiflora species identification (Mader et al. 2010). The large number of Parsimony Informative Characters (PICs) in the ITS matrix (Table 2) could also indicate a risk of substitution saturation of this marker, as previously reported for Passiflora (Muschner et al. 2003). The difficulty in obtaining high-quality ITS sequence data in Passiflora, requiring non-standard techniques to overcome a strong intramolecular secondary structure, may impair the wide adoption of the marker and increase the likelihood of miscalled bases, contributing to species mis-identification. Apart from intraspecific variation, differences in data quality are also possibly responsible for several small variances in clustering between our new sequences and those of earlier studies in certain species. This is exemplified by the variation in the ITS tree of P. rhamnifolia and P. haematostigma accessions. Variance in the chloroplast tree is likely to also result from differences in representation among the four markers in the combined matrix. The only solution to the latter problem is to assess each gene tree separately. Despite differences in gene representation between ITS and the four chloroplast markers, we obtained a gene concordance factor (gCF) of 68.6% averaged over all nodes of the ITS and combined chloroplast trees under the ML criterion (Minh et al. 2020), representing a good agreement. The mean site concordance factor (sCF) was 36.4 and mean ultrafast bootstrap (1000 pseudoreplicates) 85.9%. Due to the greater support for internal nodes, we used the combined chloroplast marker tree for interpretation of the hybridization data.

Several taxonomic questions related to the identity of the studied germplasm bank accessions were successfully resolved in this study. Among these accessions, a typical yellow-skinned sour passionfruit (P48/CPAC-MJ-M-01) clustered together with a purple-skinned accession (P46/CPAC-MJ-21-03) and another (P47/CPAC-MJ-M-23), with an unusual variation found in P. edulis: the presence of four stigmas. The ITS tree, however, distinguished the purple-skinned accession P46/CPAC-MJ-21-03 from the other studied P. edulis accessions. This is promising with a view to further investigations, with the inclusion of a larger sample of the various sub-categories of P. edulis, P. edulis f. flavicarpa and wild P. edulis Sims accessions. Significant morphological variation was detected between accessions classified as Passiflora amethystina (P. amethystina “verdadeiro”, P. amethystina “SP” and P. amethystina “rui”). An analysis of these accessions based on ITS and chloroplast DNA sequences (Figures 1 and 2) showed that P. amethystina "verdadeiro" and P. amethystina "SP" are closely related, while P. amethystina "rui" is somewhat distinct. In this context, a new analysis of the taxonomy of P. amethystina "rui" is warranted, considering its morphological differences from other accessions assigned to the species.

After repeated manual pollination attempts under controlled greenhouse conditions, 24 interspecific hybridizations were considered compatible and 11, incompatible (Table 3). Based on their placement in the combined chloroplast Bayesian tree (Figure 2), all crosses of this study between species of the subgenera Astrophea and Passiflora were incompatible, although not all possible taxon combinations were exhaustively tested. Within the same subgenus, however, many crosses were successful, whereas compatibility varied according to each subclade. For instance, all crosses between species of the same subclade within subgenus Decaloba (Figure 2, Table 3) produced viable seeds (e.g. P. sanguinolenta x P. citrina; P. sanguinolenta x P. capsularis). The same was observed for subgenus Passiflora, where all five crosses within a subclade were compatible (e.g. subclade 1a: P. laurifolia x P. nitida; subclade 1b: P. sidifolia x P. actinia). No clear cutoff value of K2P distance to predict the success or failure of Passiflora interspecific crosses could be established (Table 3). An extreme example is the short distance between the incompatible P. edulis and P. actínia and, at the other extreme, compatibility between the distantly related P. setacea and P. amethystina. Viable plants were also obtained from hybridizations between species of different subclades (Figure 2, Table 3), e.g. 1a x 1b (P. galbana x P. actinia; P. coccinea x P. actinia) and 1a x 1c (P. edulis x P. setacea; P. coccinea x P. setacea; P. setacea x P. edulis). Crosses between species of the subgroups 1d and 1a produced no fruits or viable seeds (e.g. P. serratodigitata x P. edulis; P. serratodigitata x P. coccinea). Finally, the results of crosses between species of subclades 1 and 2 were mixed, indicating compatibility of seven combinations (P. galbana x P. alata; P. galbana x P. edulis; P. quadrangularis x P. alata; P. edulis x P. caerulea; P. mucronata x P. alata, P. setacea x P. alata; P. setacea x P. amethystine) and no viable seed set of three others (P. edulis x P. tenuifila, P. caerulea x P. edulis, P. amethystina x P. edulis, P. serratodigitata x P. alata). Elsewhere, successful crosses displaying normal meiotic behavior have been achieved between P. coccinea and P. hatschbachii (Souza et al. 2020).

Some crosses were only successful in one direction, as for example P. caerulea x P. edulis, for which compatibility was only observed in one direction of the cross (P. edulis x P. caerulea). On the other hand, compatibility was confirmed for both P. edulis x P. setacea and the reciprocal (Table 3). Interspecific crosses were likely to be compatible up to an average genetic distance threshold of 0.01065, but unlikely to be successful if the genetic distance exceeded 0.01385. Species with intermediate genetic distance were identified that could serve as candidates for future bridge-cross projects with currently available fertile hybrids to motivate breeders to overcome barriers to wide crosses in this genus. In our Passiflora hybridization experiments, lower K2P distance values were somewhat predictive of compatibility (Table 3), but rather inconsistent. All crosses with K2P distances of 0.0163 and below were compatible and the largest compatible distance was 0.0247 (P. setacea x P. amethystina). Notwithstanding the crudeness of distance-based methods for phylogenetic inference compared to cladistic methods, some of the observed inconsistencies in compatibility clearly indicated that more complex and specific biological mechanisms are possibly involved in many of the interactions, rather than merely phylogenetic distances between the crossed species.

The factors affecting compatibility of interspecific hybridization are manifold and have a fundamental influence on speciation (Jiggins 2019). Some mechanisms occur prior to fertilization, preventing pollen penetration of the ovule. Post-fertilization mechanisms may include mitotic incompatibility, endosperm degeneration or differences in chromosome number (Hansen et al. 2006). In this study, incompatible crosses were detected between species with different chromosome numbers (P. mansoi x P. caerulea, P. hematoestigma x P. edulis, P. mansoi x P. edulis). However, eight other crosses between species with the same chromosome number (2n=18) were also incompatible (Table 3). The use of interspecific hybrids between two species to facilitate gene introgression with a third species that would otherwise be incompatible (bridge cross), should also be explored in more detail in interspecific Passiflora breeding (Ocampo et al. 2016).

In Brazil, Passiflora species other than P. edulis and P. alata are being cultivated and used locally for fruit consumption or pharmacological and ornamental purposes. These species include, for instance, P. cincinnata, P. nitida, P. quadrangularis, and P. setacea, which are promising candidates for intraspecific domestication and breeding, as well as for introgression of important traits into commercial passionfruit species. The interspecific crosses involving P. nitida (P. laurifolia x P. nitida), P. quadrangularis (P. quadrangularis x P. alata) and P. setacea (P. coccinea x P. setacea; P. edulis x P. setacea; P. setacea x P. alata; P. setacea x P. amethystina) described here were all compatible.

Our data suggest that phylogenetic inference could be exploited, to a certain extent, to predict the compatibility of interspecific crosses of passionfruit for breeding. This is particularly important for this genus with more than 500 species, where interspecific crosses are a common breeding technique. In breeding programs, DNA barcoding to confirm species identity and phylogenetic placement could be used as a first proxy for the selection of interspecific crosses.

REFERENCES

• Cerqueira-Silva CBM, Faleiro FG, Jesus ON, Santos ESL and Souza AP (2016) The genetic diversity, conservation, and use of passion fruit (Passiflora spp.). In Ahuja M and Jain S (eds) Genetic diversity and erosion in plants. Sustainable development and biodiversity. Springer, Cham, p. 215-231.
• Cerqueira-Silva CBM, Faleiro FG, Jesus ON, Santos ESL and Souza AP (2018) Passion fruit (Passiflora spp.) breeding. In Al-Khayri J, Jain S and Johnson D (eds) Advances in plant breeding strategies: Fruits. Springer, Cham , p. 929-951.
• Chapman MA and Burke JM (2007) Genetic divergence and hybrid speciation. Evolution 61: 1773-1780.
• Costa JL, Jesus ON, Oliveira GAF and Oliveira EJ (2012) Effect of selection on genetic variability in yellow passion fruit. Crop Breeding and Applied Biotechnology 12: 253-260.
• Dierick H, Stul M, De Kelver W, Marynen P and Cassiman J-J (1993) Incorporation of dITP or 7-deaza dGTP during PCR improves sequencing of the product. Nucleic Acids Research 21: 4427-4428.
• Feuillet C and MacDougal JM (2003) A new infrageneric classification of Passiflora L. (Passifloraceae). Passiflora 13: 34-38.
• Giudicelli GC, Mäder G and Freitas LB (2015) Efficiency of ITS sequences for DNA barcoding in Passiflora (Passiflora ceae). International Journal of Molecular Sciences 16: 7289-7303.
• Grisi MCM, Faleiro FG, Junqueira NTV and Oliveira JS (2019) Genetic variability of passion fruit multispecific hybrids and their respective wild parents determined by microsatellite markers. Journal of Agricultural Science 11: 302.
• Hansen AK, Gilbert LE, Simpson BB, Downie SR, Cervi AC and Jansen RK (2006) Phylogenetic relationships and chromosome number evolution in Passiflora . Systematic Botany 31: 138-150.
• Inglis PW, Mata LR, Silva MJ, Vieira RF, Alves RDB, Silva DB and Azevedo VC (2018b) DNA barcoding for the identification of Phyllanthus taxa used medicinally in Brazil. Planta Medica 84: 1300-1310.
• Inglis PW, Pappas MCR, Resende LV and Grattapaglia D (2018a) Fast and inexpensive protocols for consistent extraction of high-quality DNA and RNA from challenging plant and fungal samples for high throughput SNP genotyping and sequencing applications. PLoS One 13: e0206085.
• Jiggins CD (2019) Can genomics shed light on the origin of species? PLoS Biology 17: e3000394.
• Krosnick SE, Porter-Utley KE, MacDougal JM, Jørgensen PM and McDade LA (2013) New insights into the evolution of Passiflora subgenus Decaloba (Passiflora ceae): phylogenetic relationships and morphological synapomorphies. Systematic Botany 38: 692-713.
• Kumar S, Stecher G and Tamura K (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular Biology and Evolution 33: 863-869.
• Lira Júnior JS, Flores PS and Bruckner CH (2014) UFV-M7: mutant yellow passionfruit genotype with photoperiod insensitivity for flowering. Crop Breeding and Applied Biotechnology 14: 128-131.
• MacDougal JM and Feuillet C (2004) Systematics. In Ulmer T and MacDougal JM (eds) Passiflora : Passionflowers of the world. Timber Press, Portland, p. 27-31.
• Mader G, Zamberlan PM, Fagundes NJR, Magnus T, Salzano FM, Bonatto SL and Freitas LB (2010) The use and limits of ITS data in the analysis of intraspecific variation in Passiflora . L. (Passiflora ceae). Genetics and Molecular Biology 33: 99-108.
• Mezzonato-Pires AC, Milward MA, Mendonça CBF and Gonçalves-Esteves V (2018) Taxonomy, palynology and distribution notes of seven new records of Passiflora L. (Passiflora ceae s.s.) for Brazil. PhytoKeys 95: 1-14.
• Minh BQ, Hahn MW and Lanfear R (2020) New methods to calculate concordance factors for phylogenomic datasets. Molecular Biology and Evolution 37: 2727-2733.
• Muñoz-Sanz JV, Zuriaga E, Cruz-García F, McClure B and Romero C (2020) Self-(In)compatibility systems: target traits for crop-production, plant breeding, and biotechnology. Frontiers in Plant Science 11: 195.
• Muschner VC, Lorenz AP, Cervi AC, Bonatto SL, Souza-Chies TT, Salzano FM and Freitas LB (2003) A first molecular phylogenetic analysis of Passiflora (Passiflora cae). American Journal of Botany 90: 1229-1238.
• Muschner VC, Zamberlan PM, Bonatto SL and Freitas LB (2013) Phylogeny, biogeography and divergence times in Passiflora (Passiflora ceae). Genetics and Molecular Biology 35: 1036-1043.
• Ocampo J, Arias J and Urrea R (2016) Interspecific hybridization between cultivated and wild species of genus Passiflora L. Euphytica 209: 395-408.
• Ramaiya SD, Bujang JS and Zakaria MH (2014) Genetic diversity in Passiflora species assessed by morphological and ITS sequence analysis. The Scientific World Journal 2014: e598313.
• Ronquist F, Teslenko M, Van Der Mark P, Ayres DL, Darling A, Höhna S, Larget B, Liu L, Suchard MA and Huelsenbeck JP (2012) MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology 61: 539-542.
• Souza VO, Souza MM, Silva GS, Melo CAF, Freitas JCO and Souza AC (2020). Morphological and cytogenetic characterization of new ornamental Passiflora hybrids (P. ‘Vivis’ and P. ‘Jhovi’). Crop Breeding and Applied Biotechnology 20: e276920313.
• Swofford DL (2003) PAUP*. phylogenetic analysis using parsimony (* and other methods). version 4. Sinauer Associates, Sunderland, Massachusetts, 142p

Publication Dates

• Publication in this collection
  21 May 2021
• Date of issue
  2021

History

• Received
  17 Dec 2020
• Accepted
  04 Mar 2021
• Published
  10 May 2021