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Phylogeny and historical biogeography analysis support Caucasian and Mediterranean centres of origin of key holoparasitic Orobancheae (Orobanchaceae) lineages
expand article infoRenata Piwowarczyk, Adam C. Schneider§, Grzegorz Góralski|, Dagmara Kwolek|, Magdalena Denysenko-Bennett|, Anna Burda|, Karolina Ruraż, Andrzej J. Joachimiak|, Óscar Sánchez Pedraja
‡ Jan Kochanowski University, Kielce, Poland
§ Department of Biology and Health Sciences, Hendrix College, Conway, United States of America
| Jagiellonian University, Kraków, Poland
¶ Grupo Botánico Cantábrico, Liérganes, Spain
Open Access

Abstract

The extensive diversity of the tribe Orobancheae, the most species-rich lineage of holoparasitic Orobanchaceae, is concentrated in the Caucasus and Mediterranean regions of the Old World. This extant diversity has inspired hypotheses that these regions are also centres of origin of its key lineages, however the ability to test hypotheses has been limited by a lack of sampling and phylogenetic information about the species, especially in the Caucasus region. First, we assessed the phylogenetic relationships of several poorly known, problematic, or newly described species and host-races of four genera of Orobancheae occurring in the Caucasus region–Cistanche, Phelypaea, Phelipanche and Orobanche–using nuclear ribosomal (ITS) and plastid (trnL–trnF) sequence data. Then we applied a probablistic dispersal-extinction-cladogenesis model of historical biogeography across a more inclusive clade of holoparasites, to explicitly test hypotheses of Orobancheae diversification and historical biogeography shifts. In sum, we sampled 548 sequences (including 196 newly generated) from 13 genera, 140 species, and 175 taxa across 44 countries. We find that the Western Asia (particularly the Caucasus) and the Mediterranean are the centre of origin for large clades of holoparasitic Orobancheae within the last 6 million years. In the Caucasus, the centres of diversity are composed both of long-branch taxa and shallow, recently diversified clades, while Orobancheae diversity in the Mediterranean appears to represent mainly recent diversification.

Keywords

Biodiversity hotspot, chronogram, Cistanche, divergence time, historical biogeography, Orobanche, Phelipanche, Phelypaea

Introduction

The tribe Orobancheae is the oldest and most species-rich of the three lineages of holoparasites comprising the cosmopolitan family Orobanchaceae, with a crown age dating to the mid-Miocene (McNeal et al. 2013; Schneider and Moore 2017). In its current circumscription the Orobanchaceae includes the holoparasites that have always comprised Orobanchaceae s. str., and hemiparasites traditionally included in Scrophulariaceae (Olmstead et al. 2001; McNeal et al. 2013). In sum, this is the largest parasitic plant family with 102 genera and over 2,100 species (Nickrent 2020) which together with its variety of trophic modes makes it a valuable model for studying the evolution and physiology of parasitism (Westwood et al. 2010).

The Mediterranean Basin and Caucasus region of western Asia are centres of extant diversity for the two most diverse genera in the Orobancheae, Orobanche L. and Phelipanche Pomel (ca. 150 and 60 described species, respectively) (Piwowarczyk et al. 2019), and are more generally recognised as one of the world’s hotspots of biodiversity (Mittermeier et al. 2005). Recent taxonomic and field studies in the Caucasus have helped clarify the nomenclature, taxonomy, and distribution of taxa from four genera (Orobanche L., Phelipanche Pomel, Phelypaea L. (= Diphelypaea Nicolson) and Cistanche Hoffmannsegg & Link), and revealed many endemic and host-specific species in this region that had previously been overlooked (e.g., Piwowarczyk 2015; Piwowarczyk et al. 2015, 2017a, b, c, d, 2018a, b, c, 2019, 2020a, 2021). Other researchers have refined the understanding of these four genera in the Mediterranean Basin, as well as the monotypic Boulardia F.W. Schultz. (e.g., Foley 2001; Carlón et al. 2003, 2005, 2008; Domina and Arrigoni 2007; Jeanmonod and Habashi 2007; Pujadas Salvà 2009; Domina et al. 2011, 2013; Frajman et al. 2013). Together, the Mediterranean and the Caucasus have been hypothesised as refugia for both plant and animal lineages during the Pleistocene ice ages (Taberlet et al. 1998; Hewitt 1999; Lumibao et al. 2017), and some authors even propose the Caucasus together with the Middle East and Central Asian high mountains as the main area of origin of Old World broomrapes (Orobanche and Phelipanche, Rätzel and Uhlich 2004).

While regions of high extant diversity for any lineage may be the result of in situ diversification, this is not necessarily the case. Thus, hypotheses of historical biogeography must be explicitly tested. Schneider and Moore (2017) used a statistical phylogenetic framework to infer the divergence times and historical biogeography of the Orobancheae to the extent possible given the limitations of a depauperate fossil record and the increased rates of molecular evolution that are characteristic of parasitic plants (Bromham et al. 2013). While an important first step, their study focused on New World taxa and therefore lacked the taxonomic sampling or granularity of geographical data to evaluate biogeographical patterns within the Old World.

The aims of this study were two-fold. First, we sought to assess previously unknown phylogenetic relationships of Caucasian Orobancheae using nuclear ribosomal (ITS region) and plastid (trnL–trnF) DNA sequences. Second, we sought to evaluate the historical biogeography of Old World Orobancheae using a probabilistic dispersal-extinction-cladogenesis (DEC) model. In particular, we wanted to evaluate the hypothesis of Western Asia (especially the Caucasus) and the Mediterranean as potential refugia and/or centres of origin for major species-rich clade in the Orobancheae.

Materials and methods

Taxonomic sampling and data collection

For the initial phylogenetic analysis, we studied Caucasian species of Cistanche, Phelypaea, Phelipanche and Orobanche, mainly collected from Georgia, Armenia, Azerbaijan and Russia between 2014 and 2019. Specimens of some species were collected in other countries or taken from herbaria (B, ERCB, HMMNH, IRKU, KTC, LE, MW, herb. Ó. Sánchez Pedraja), or sequences were downloaded from GenBank. In total, 13 genera, 175 taxa representing 140 species (548 sequences, including 196 as new), from 44 countries, were analysed (see Suppl. material 1: Table S1). For the majority of samples, hosts were precisely identified. We assessed infraspecific variation by sampling more than one individual, often from different localities and host species. Voucher information, as well as geographic origin or GenBank accession numbers are listed in Suppl. material 1: Table S1. Newly collected plant specimens were deposited in KTC, ERCB (herbarium codes according to Index Herbariorum, Thiers 2017). Systematic division was adopted according to Beck (1930) and Teryokhin et al. (1993), the scheme followed explicitly or implicitly by most researchers, and some recent taxonomic changes made by Piwowarczyk et al. (2017a, 2018d, 2019) (Fig. 1).

Figure 1. 

Rooted Maximum Likelihood phylogenetic tree constructed using ITS sequences. Numbers near branches show ultrafast bootstrap values (values ≥ 75 are shown). The bar represents the amount of genetic change (nucleotide substitutions per site) A summary of backbone (generic) relationships, branches connecting the outgroup Lindenbergia and Boulardia are shortened to fit the figure B–E relationships of taxa within the genera Cistanche, Phelipanche, Phelypaea, and Orobanche respectively. Species names, the country of origin, host species (if available) and GenBank number are included on the phylogeny tip labels.

Figure 1. 

Continued.

Material used for DNA extraction was freshly collected and silica gel-dried or was obtained from herbarium vouchers. For phylogenetic studies we used two types of sequences: nuclear ITS region (internal transcribed spacer 1, 5.8S ribosomal RNA gene, internal transcribed spacer 2, later referred to as ITS) and plastid trnL–trnF sequence (RNA-Leu (trnL) intron, the partial trnL gene, and the intergenic spacer between the trnL 3’ exon and tRNA-Phe (trnF) gene region’s plastid DNA). These two regions are commonly used for species-level phylogenetic inference, including in the Orobancheae (ITS: Schneeweiss et al. 2004; Carlón et al. 2005, 2008; Park et al. 2008; Schneider et al. 2016; Fu et al. 2017; Piwowarczyk et al. 2018d; trnL–trnF: Schneider et al. 2016; Piwowarczyk et al. 2018d). DNA extraction and sequence amplification procedures follow the methods of Piwowarczyk et al. (2018d).

Phylogenetic inference

Sequences were aligned with MAFFT v7.407 (Katoh and Standley 2013), manually corrected and trimmed. The final number of sequences and length of alignments were: for ITS 229 sequences of 671 positions, in the case of trnL–trnF 153 sequences, 1,337 positions long. Separate ITS and trnL–trnF trees were inferred instead of concatenating them into a single analysis for two main reasons: first, although preliminary trees inferred from each sequence were generally congruent, certain species did show conflicting placements (described below), perhaps due to differences in plastid versus nuclear inheritance. Second, the ITS tree is much richer in samples, because of greater availability in GenBank. Information about sequences (newly obtained and downloaded from GenBank) used in phylogenetic analysis is presented in Suppl. material 1: Table S1.

For both sequence alignments, Maximum Likelihood (Figs 1, 2) and Bayesian (Suppl. material 2: Fig. S1, Suppl. material 3: Fig. S2) phylogenetic trees were generated with Lindenbergia sinaica (Decne.) Benth. used as outgroup. Maximum Likelihood (ML) trees were calculated with IQ-TREE multicore version 1.6.12 (Nguyen et al. 2015) software, with ultrafast bootstrap approximation (2,000 bootstrap replicates). Substitution models were auto-determined by IQ-TREE using the Bayesian Information Criterion (BIC) (SYM+I+G4 for ITS and TVM+F+R3 for trnL–trnF). Bayesian phylogenetic trees were generated using MrBayes v. 3.2.6 (Huelsenbeck et al. 2001; Ronquist and Huelsenbeck 2003) with the following main settings: ngen = 10,000,000, samplefreq = 500, nchains = 4, checkfreq = 100,000, diagnfreq = 5,000, stopval = 0.01, stoprule = yes, relburnin = yes, burninfrac = 0.25 and, lset applyto = (all) nst = 6 rates = invgamma (for ITS) or lset applyto = (all) nst = 6 rates = gamma (for trnL–trnF). Substitution models according to BIC (SYM+I+G for ITS and GTR+G for trnL–trnF) were determined by IQ-TREE software. The trees were visualised by iTOL tool (Letunic and Bork 2016).

Figure 2. 

Rooted Maximum Likelihood phylogenetic tree constructed using plastid trnL–trnF spacer sequences. As an outgroup, Lindenbergia sinaica was used. Numbers near branches show ultrafast bootstrap values (values ≥ 75 are shown). The bar represents the amount of genetic change (nucleotide substitutions per site) A summary of backbone (generic) relationships B Phelipanche clade C Orobanche clade. Species names, the country of origin, host species (if available) and GenBank number are included on the phylogeny tip labels.

Historical biogeography

To infer a chronogram for historical biogeography analysis of the tribe Orobancheae we used the ITS, PhyA, and PhyB Orobancheae alignments of Schneider and Moore (2017), improved in six ways:

1. Taxonomic coverage for Orobanche and Phelipanche was expanded based on this study.

2. Taxonomic coverage for Cistanche was expanded by using sequence data submitted to GenBank by Ataei (2017). Recent phylogenetic evidence support many more lineages in this genus than previously recognized (Ataei 2017; Ataei et al. 2020). Names for some of these lineages have been proposed but not yet validly published (Ataei 2017).

3. Sequences for Gleadovia Gamble & Prain and Phacellanthus Siebold & Zucc. – first published by Fu et al. (2017) and available on GenBank – were added, resulting in complete taxonomic coverage at the genus level, except for the monotypic Mexican genus Eremitilla Yatsk. & J.L.Contr.

4. The trnL–trnF plastid locus was added for most taxa based on newly generated data or pre-existing sequences (Suppl. material 1: Table S1). Although nrDNA and cpDNA partitions support conflicting relationships for a few taxa, the key nodes associated with major biogeographic transitions and discussed herein are supported by both analyses.

5. A 637 bp region of the PhyA gene was excluded from analysis because it was poorly alignable. This region appears only in our sequences for Boschniakia himalaica Hook. f. & Thomson ex Hook. f. and Aphyllon ludovicianum (Nutt.) A.Gray but not for any other species.

6. Samples for Aphyllon californicum (Cham. & Schltdl.) A.Gray subspecies feudgei, grande, grayanum, and jepsonii were replaced with different samples for which both ITS and trnL–trnF sequences were available.

Sequences matrices for each gene were aligned separately using Geneious 9.1.8 (Biomatters, Auckland, New Zealand; Kearse et al. 2012), then concatenated into a single supermatrix comprised of a 1986bp ITS + trnL–trnF backbone plus 3375 bp of phytochrome sequence from a subset of 20 taxa. In this case, we decided that the better branchlength estimates broadly across the tree by using multiple genes generally outweighed errors introduced for particular tips that may have conflicting ITS and trnL–trnF topologies. This supermatrix was used to infer a chronogram by implementing an uncorrelated lognormal relaxed clock model and a GTR+Γ substitution model in the software Revbayes v. 1.0.11 (Höhna et al. 2014). Our starting tree was generated using default parameters in RAxML-HPC v8, run on XSEDE through the CIPRES portal (Stamatakis 2014), rooted based on the results of previous comparable molecular phylogenetic studies (McNeal et al. 2013; Fu et al. 2017; Schneider and Moore 2017), and made ultrametric with a root age set to 25 (Ma) using the rate-smoothing function chronos in the R package ‘ape’ v. 5.3 (Paradis and Schliep 2019). The same divergence time calibrations and other analysis parameters were used as in Schneider and Moore (2017), except we used a new starting tree and the Markov Chain Montecarlo (MCMC) analysis was run for 4,000 iterations as a pre-burnin to tune the proposal parameters then sampled every 100 iterations for 50000 iterations with the first 15% of samples discarded as burn-in.

Each iteration consisted of 472 moves randomly scheduled from 394 possible moves. Stationarity was assessed using Tracer v.1.7.1 (Rambaut et al. 2018) and the effective sampling size of each important parameter exceeded 200: likelihood, prior, each GTR parameter and the shape parameter for the gamma distribution for each partition, speciation and extinction rates, root time, and clade ages of Orobanche s.l. and Cistanche.

For biogeographical analysis, the global range of Orobancheae was divided into six non-overlapping regions based on physical geography and natural phytogeographic divisions (Fig. 3): (1) Europe/Mediterranean, including Central, North, Eastern and Southern Europe with Mediterranean Basin (Iberian, Italian and Balkan peninsulas with northern Africa – north from Sahara Desert, and western and southern parts of the peninsula of Turkey); (2) Western Asia, which includes Anatolia in Turkey, the Arabian Peninsula, Iran, the Levant, Mesopotamia, the Sinai Peninsula, and Caucasus (with Transcaucasia); (3) Central Asia, including the area from the Caspian Sea to western China, and from Afghanistan, through Turkmenistan, Tajikistan, Uzbekistan, Kyrgyzstan, and Kazakhstan to the south to Russia (with Ciscaucasia) in the north; (4) East Asia, from central China eastward (Hong Kong, Macao, Mongolia, the Korean peninsula, Japan, and Taiwan) and including Australia for Orobanche cernua var. australiana (F. Muell.) Beck, the only taxon apparently native to that continent in our study; (5) Africa, south of the Mediterranean Basin (Saharan and sub-Saharan); and (6) the New World. In general, a taxon was not considered to inhabit a region if < 5% of its known range fell within the respective region boundary. To determine the range for individual species, we used a variety of peer-reviewed sources (e.g., Novopokrovskij and Tzvelev 1958; Wu and Raven 1998; Pusch and Günther 2009; Cullen 2010; Tzvelev 2015; Ataei 2017; Freeman et al. 2019; Piwowarczyk et al. 2019) and continually updated databases (Domina and Raab-Straube 2010; Sánchez Pedraja et al. 2016), supported by our knowledge acquired during field and herbaria research. Ataei (2017) was used to determine the distribution of undescribed Cistanche taxa who we follow along with Ataei et al. (2020) because they have the most comprehensive set of genetic data. However, some taxonomic and distributional ranges conflict with other recent treatments (Moreno Moral et al. 2018), highlighting the need for continued evaluation in this genus.

Figure 3. 

Historical biogeography of tribe Orobancheae, reconstructed using a dispersal-extinction-cladognesis model implemented in RevBayes (maximum likelihood topology, maximum clade credibility branch lengths). Coloured circles at tips represent the current biogeographical range of each sampled taxon. Circles on each node represent the reconstructed ancestral area of the most recent common ancestor of the two daughter lineages, while circles on either side of the node show the reconstructed areas immediately following cladogenesis. Circle size is proportional to posterior probability. Each colour represents a different biogeographical region or combination of regions as indicated by the map and legend to the left of the chronogram. Tip labels for Cistanche follow nomenclature of Ataei et al. (2020). Asterisks indicate names proposed by Ataei (2017) but not yet validly published.

Ancestral geographical ranges were inferred by applying a dispersal-extinction-cladogenesis (DEC) model of historical biogeography to the maximum clade credibility (MCC) tree from the Bayesian analysis. The DEC model, also implemented in RevBayes, allows for sympatric speciation, allopatric speciation and anagenetic range expansion and contraction (Ree and Smith 2008). Two independent MCMC replicates were run for 1,000 iterations as a pre-burn-in to tune the proposal settings, then sampled every 5 iterations for 10000 iterations. Each iteration consisted of 11 moves randomly scheduled from 3 possible moves. Stationarity was also assessed using Tracer.

Results

Phylogenetic relationships

The most important results of our phylogenetic analyses clarified the position of many previously unsampled Caucasian species (Figs 1, 2, Suppl. material 2: Fig. S1, Suppl. material 3: Fig. S2). We also showed the phylogenetic relations of the newly described species, i.e., Phelipanche zangezuri Piwow. et al., P. hajastanica Piwow. et al., and P. sevanensis Piwow. et al., O. javakhetica Piwow. et al., O. arpica Piwow. et al. and O. zajaciorum Piwow.

Consistent with previous studies, the studied genera were each strongly supported as monophyletic (Bootstrap (BS) ≥ 90, Posterior Probability (PP) = 1.0).

Cistanche

ITS (trnL–trnF data was not available) trees show that Cistanche armena (K. Koch) M.V. Agab. (samples from two different hosts, Alhagi Gagnebin and Salsola L.) is closely related to C. deserticola Ma and C. salsa (C.A. Mey.) Beck (BS = 100, PP = 1.00), and with the later one it has sometimes been confused (Fig. 1, Suppl. material 2: Fig. S1).

Phelypaea

The three species from genus Phelypaea, P. tournefortii Desf. and P. coccinea (M. Bieb.) Poir. are clearly separated (BS = 100, PP = 1.00), however P. boissieri (Reut.) Stapf, first sequenced for this study, seems to be very similar to P. coccinea. Amplification of trnL–trnF in Phelypaea samples was successful only in the case of P. coccinea, so the above analysis was based only on ITS (Fig. 1, Suppl. material 2: Fig. S1).

Phelipanche

Based on ITS data P. zangezuri is separated from the clade of P. caesia (Rchb.) Soják (BS = 97, PP = 0.90) and the clade containing remain Phelipanche species (BS = 98, PP = 0.85). By contrast, trnL–trnF trees do not indicate separation of P. zangezuri and P. caesia. Rather, samples of P. arenaria form a sister clade to these two species, and together form a well-supported lineage (BS = 98, PP = 1.00) separated from the rest of Phelipanche (BS = 95, PP = 0.96) (Figs 1, 2, Suppl. material 2: Fig. S1, Suppl. material 3: Fig. S2). P. sevanensis is closely related to the group of P. schultzii (Mutel) Pomel and P. heldreichii (Reut.) Soják on all trees, and to P. cernua Pomel. on the ITS trees, (BS = 99, PP = 0.99) (Figs 1, 2, Suppl. material 2: Fig. S1, Suppl. material 3: Fig. S2). However, P. hajastanica is found in the group of slightly differentiable species on the ITS tree (Fig. 1), while on the trnF-trnL tree it is close to P. cilicica (Beck) Soják (BS = 99, PP = 0.92) (Fig. 2).

Our results showed the relationship of samples from different parts of the range of disjunctive species, such as P. portoilicitana (A. Pujadas & M.B. Crespo) Carlón et al. and P. cernua. Whereas trnL–trnF sequences of P. cernua places samples from Armenia and Spain are grouped in the same clade (BS = 98, PP = 0.94), on the ITS tree, the European samples are separated from Caucasian sample which is in the same clade as P. sevanensis, P. schultzii and P. heldreichii (BS = 99, PP = 0.99). Also, P. portoilicitana, both on ITS and trnL–trnF trees, show differences between samples from Armenia and Spain (Figs 1, 2, Suppl. material 3: Fig. S2).

Orobanche

Orobanche gamosepala Reut. is genetically distinct (BS = 100, PP = 1.00) from O. anatolica Boiss. & Reut. ex Reut./O. colorata K. Koch and together these species are grouped in sister clade to the rest of Orobanche species (ITS: BS = 99, PP = 1.00, trnL–trnF: BS = 100, PP = 1.00) (Figs 1, 2, Suppl. material 2: Fig. S1, Suppl. material 3: Fig. S2).

ITS sequence data indicates that O. cicerbitae (Uhlich & Rätzel) Tzvelev is not closely related to O. flava Mart. ex F.W. Schultz, however on the trnL–trnF trees O. cicerbitae from Georgia and Azerbaijan forms a common clade with O. flava from Georgia (BS = 98, PP = 0.97), whereas Central European samples of O. flava are distant (Fig. 1, Suppl. material 2: Fig. S1).

ITS sequences (Fig. 1, Suppl. material 2: Fig. S1) of high mountain Orobanche species, such as O. krylowii Beck, O. cicerbitae, O. arpica, O. mlokosiewiczii Piwow. et al., O. inulae Novopokr. & Abramov and O. lycoctoni Rhiner showed that they are closely related, and form a separated clade (ITS: BS = 100, PP = 1.00, trnL–trnF: BS = 99, PP = 1.00) included in O. ser. Krylowianae Piwow. et al. Probably these species diverged relatively recently and can be an example of recent rapid radiation. Another interesting phenomenon is the placement of the trnL–trnF sequence (Fig. 2, Suppl. material 3: Fig. S2) of O. lycoctoni on phylogenetic trees near O. lucorum A. Braun ex F.W. Schultz, (BS = 99, PP = 1.00), a species distantly related to the sect. Krylowianae species.

The phylogenetic position of Caucasian endemic species with unclear affinity has also been presented, in particular those previously classified in inappropriate subsections, such as O. schelkownikovii Tzvel., O. grossheimii Novopokr., O. raddeana Beck, and O. laxissima Rätzel & Uhlich (Figs 1, 2, Suppl. material 2: Fig. S1, Suppl. material 3: Fig. S2, and discussion below).

Little within-species variation is shown among the samples from different host species taken from the following species: O. laxissima, O. alba Stephan ex Willd., O. bartlingii Griseb., O. caryophyllacea Sm., O. cicerbitae, O. gracilis Sm., O. centaurina Bertol., O. minor Sm., O. owerinii (Beck) Beck, O. raddeana, O. schelkovnikovii, P. cilicica, P. coelestis (Reut.) Soják, P. purpurea (Jacq.) Soják and P. coccinea (Figs 1, 2, Suppl. material 2: Fig. S1, Suppl. material 3: Fig. S2).

Historical biogeography

We find negligible support (PP < 0.4) for any single hypothesis ancestral range of lineages older than 6 million years. However, most diversification in the Orobancheae has happened relatively recently (Tables 1, 2, Fig. 3). We focus below on Cistanche, Phelipanche and Orobanche because these are the three most diverse lineages in the Old World and we found relatively high support for some biogeographical patterns. Phelypaea probably originated in West Asia (PP = 0.46) or was more widespread in West Asia and Caucasus, Europe and the Mediterranean (PP = 0.11).

Table 1.

Divergence times with credible intervals (95% highest probability density (HPD)) and inferred historical biogeography of selected clades. Biogeographical regions defined in Methods and Figure 3.

Clade Crown Age (Ma) Biogeography
Mean 95% HPD Region Posterior Prob.
Cistanche sect. Heterocalyx 3.0 2.0–4.2 Central Asia 0.28 (0.42 for clade excluding C. deserticola)
Western Asia 0.14 (0.27)
Phelypaea 2.4 1.4–3.7 Western Asia 0.46
Western Asia + Med/Europe 0.11
Phelipanche 3.9 2.8–5.5 Western Asia 0.41
Europe/Mediterranean 0.09
Both 0.11
Phelipanche clade P1 1.8 1.1–2.4 Western Asia 0.38
Western Asia + Med/Europe 0.32
Phelipanche clade P2 0.753 0.52–1.1 Western Asia 0.58
Western Asia + Europe/Med 0.34
Orobanche clade O1 0.44 0.26–0.67 Europe/Mediterranean 0.93
Orobanche clade O2 0.72 0.46–1.0 Europe/Mediterranean 0.99
Orobanche clade O1+O2 0.81 0.52–1.1 Western Asia + Europe/Med 0.50
Europe/Mediterranean 0.24
Orobanche clade O3 1.27 0.75–1.8 Western Asia 0.93
Orobanche clade O4 0.75 0.40–1.1 Europe/Mediterranean + Western Asia n/a
+ Central Asia 0.12
+ East Asia 0.10
+ Both 0.27
Table 2.

Divergence times of species or clades endemic or nearly endemic to the Caucasus region.

Species or clade, or paraphyletic groupa Taxa Divergence timea (Ma) 95% HPD
Clade Phelypaea coccinea + P. tournefortii 2.4 1.4–3.7
Clade Orobanche anatolica + O. colorata + O. gamosepala 5.4 3.5–7.6
Paraphyletic Orobanche arpica, O. cicerbitae on Caucasalia, O. cicerbitae, O. inulae, O. mlokosiewiczii, O. cicerbitae on Pojarkovia (+ widespread O. krylowii) 1.3 0.75–1.8
Species Orobanche zajaciorum 1.7 1.2–2.3
Species Orobanche raddeana 2.2 1.7–3.3
Species Orobanche grossheimii 1.2 0.74–1.5
Clade Orobanche laxissima + O. owerinii + O. transcaucasica 0.08 0.02–0.15
Species Orobanche javakhetica 2.4 1.6–3.2
Species Orobanche kurdica 0.08 0.0002–0.23
Species Orobanche schelkovnikovii 0.50 0.30– 0.69
Species Phelipanche bungeana 1.48 0.93–2.0
Clade Phelipanche coelestis + Phelipanche “on Astrodaucus 0.15 0.05–0.26
Species Phelipanche hajastanica 0.28 0.16–0.40
Clade Phelipanche heldreichii + P. sevanensis 0.19 0.08–0.32
Species Phelipanche “on Artemisia 1.1 0.52–1.8
Species Phelipanche “on Genista 0.20 0.10–0.31
Species Phelipanche pulchella 0.41 0.21–0.61
Species Phelipanche zangezuri 0.68 0.33–1.1

Cistanche

The phylogeny of Cistanche appears to be structured by geography, with clades of species endemic to particular areas. For example, we find weak support for a Central Asian ancestor of Cistanche sect. Heterocalyx sensu Ataei, non Beck (composed of C. salsa, C. bamianica Ataei ined. (Ataei 2017), C. bilobata Ataei ined. (Ataei 2017), C. deserticola, C. ambigua (Bunge) Beck (= C. trivalvis (Trautv.) Korsh.), C. tomentosa Ataei ined. (Ataei 2017), C. ridgewayana Aitch. & Hemsl. and C. persica Ataei ined. (Ataei 2017)) (PP = 0.28; 0.42 for the subclade excluding the more widespread C. deserticola). Several extant species such as C. persica, C. tomentosa, and C. salsa extend further west into the Europe/Mediterranean region; we inferred that these are the result of recent range expansions (Fig. 3).

We found support that the clade of species C. algeriensis Ataei ined. (Ataei 2017), C. almeriensis Ataei ined. (Ataei 2017), C. phelypaea (L.) Cout. and C. violacea (Desf.) Hoffmanns. & Link in Cistanche sect. Cistanche sensu Ataei (= Cistanche sect. Eucistanche Beck, p.p. max.) originated from an ancestor that was either widespread throughout the European/Mediterranean region and Western Asia (PP = 0.47), or just restricted to Europe/the Mediterranean (PP = 0.44). We also inferred a Western Asian origin for the clade of species C. chabaharensis Ataei ined. (Ataei 2017), C. tubulosa (Schenk) Hook. f., C. senegalensis (Reut.) Beck, C. laxiflora Aitch. & Hemsl., and C. flava (C.A. Mey.) Korsh. (PP = 0.95), although extant species are found throughout Western and Central Asia today.

Phelipanche

We found moderate support for a Western Asia origin of Phelipanche (PP = 0.41) approximately 2.8–5.5 million years ago, with alternative biogeographical hypotheses much more weakly supported (Table 1). Within the genus we found strong support for two general observations. First, important subclades of Phelipanche also likely originated in Western Asia or were more widespread into Europe or the Mediterranean as well. These include the large subclades designated P1 and P2 in Figure 3, the crown ancestors of which were most probably limited to Western Asia (PP = 0.38 and 0.58, respectively), but may have had a larger range extending into Europe/the Mediterranean Basin as well (PP = 0.32 and 0.34). Crown ancestors of clades nested within P2 were inferred to be limited Western Asia with even higher probability (PP > 0.7) with dispersal out of this region by some extant species (e.g., P. gratiosa (Webb) Carlón et al. (Canary Islands, endemic) to the Mediterranean & Europe, as well as the subclade P. libanotica (Schweinf. ex Boiss.) Soják + P. reuteriana (Rchb. fil.) Carlón et al. + P. oxyloba (Reut.) Soják + P. georgii-reuteri Carlón et al. + P. cilicica + P. aegyptiaca (Pers.) Pomel), and independent range expansions into Europe/the Mediterranean Basin from an ancestor limited to Western Asia in the sister species P. lavandulacea (Rchb.) Pomel and P. mutelii (F.W. Schultz) Pomel (PP ≥ 0.75).

Similarly, we find it most probable that the most widespread and often weedy species of Phelipanche had direct stem ancestors limited in range to Western Asia. These include Phelipanche arenaria (Borkh.) Pomel (PP = 0.71), P. caesia (PP = 0.5), P. ramosa (L.) Pomel (PP = 0.90), and P. aegyptiaca (PP = 0.40, with the next most probable origin as Europe/the Mediterranean, PP = 0.22).

Orobanche

Similar to Phelipanche we infer a Western Asian origin for ancestral Orobanche (PP = 0.43; 0.39 for Orobanche + Boulardia). Four key subclades are diagnosable by their biogeographic affinities. The first and second subclades are closely related and comprise predominantly Europe/Mediterranean species that have diversified in situ (O1 + O2 in Table 1, Fig. 3; ancestral range Europe/Mediterranean, PP = 0.93 and 0.99). The larger clade, also including the widespread O. alba and the Caucasian endemic O. grossheimii, likely originated in Western Asia (PP = 0.58; Fig. 3, Suppl. material 4: data S1 and Suppl. material 5: data S2) or Western Asia + Europe/the Mediterranean (PP = 0.24). A Western Asian origin is even more probable for the several more inclusive clades of Orobanche moving toward the root (PP = 0.66 – 0.84). The third key clade, O3, is composed exclusively of Caucasian endemics, except for the more widespread species O. krylowii (indicated as O3 in Fig. 3) and also originated in Western Asia (PP = 0.93, Tables 1, 2). Finally, the fourth clade (O4) consists of several widespread species including O. cernua L., O. amoena C.A. Mey., O. cumana Wallr., O. pycnostachya Hance, and O. grenieri F.W. Schultz and was also inferred to have a widespread common ancestor, though the exact geography is uncertain (Table 1). The top three most probable biogeographic states for the common ancestor encompass the regions Europe/Mediterranean plus Western Asia as well as either Central Asia, Eastern Asia, or both, but together these hypotheses only represent half of the posterior density (Table 1).

Discussion

Phylogenetic relationships

Cistanche

C. armena was described by Koch (1843) as Phelypaea armena, synonymised with P. salsa C.A. Mey by Boissier (1879) and transferred to C. salsa by Beck, where it has remained in synonym by subsequent authors (Ataei 2017; Ataei et al. 2020). However, recent morphological study has indeed shown that C. armena differs clearly from C. salsa (Piwowarczyk et al. 2019). The occurrence of this Cistanche species in the Caucasus requires further field and molecular studies, however at this point C. armena is known only from Armenia, and C. salsa and C. fissa (C.A. Mey.) Beck probably are absent from Armenia or Georgia (Piwowarczyk et al. 2019).

Phelypaea

This genus includes three holoparasite species (P. coccinea, P. boissieri, and P. tournefortii) that parasitize Asteraceae hosts. Phelypaea coccinea, a parasite of Psephellus Cass. and Centaurea L., rarely Klasea Cass., occurs in the Caucasus and Crimea, while P. tournefortii, a parasite of Tanacetum L., occurs in the Caucasus and Turkey (Piwowarczyk et al. 2019). However, P. boissieri shows a different distribution; it occurs in the Balkans (Albania, Greece, North Macedonia), and Western Asia (Turkey, Iraq and Iran), parasitises Centaurea (similarly P. coccinea), and occasionally Cousinia Cass. in Iraq (Piwowarczyk et al. 2019). The molecular (Fig. 1) and morphological features that separate P. coccinea and P. boissieri – i.e., corolla-tube short and cup-shaped; corolla-lobes broadly obovate-orbicular to orbicular, overlapping; anthers hairy (Piwowarczyk et al. 2019) – are not fully differentiating. Thus, further research into variability and the inclusion of more samples for genetic analysis are required.

Phelipanche

The phylogenetic relations of the newly described species, i.e., P. zangezuri (Piwowarczyk et al. 2018a), P. hajastanica (Piwowarczyk et al. 2017c), and P. sevanensis (Piwowarczyk et al. 2017b) are presented (Figs 1, 2).

Phylogenetic analysis of two species previously known mainly from the Mediterranean area and later found in the Caucasus, i.e., P. portoilicitana and P. cernua (Piwowarczyk et al. 2019), showed some differences between samples collected from these different parts of the range. This may indicate the ongoing process of speciation despite similarity in host association (Figs 1, 2).

ITS (Fig. 1) poorly differentiates some species aggregates in Phelipanche sect. Phelipanche, while it does well in the sect. Trionychon (Wallr.) Piwow. & Ó. Sánchez (Piwowarczyk et al. 2018d, = sect. Arenariae Teryokh.).

Orobanche

The recently described O. flava subsp. cicerbitae Uhlich & Rätzel [≡ O. cicerbitae (Uhlich et Rätzel) Tzvelev] parasitising Cicerbita Wallr. and Senecio propinquus Schischk. is distantly related to O. flava, at least as far as ITS (Fig. 1, Suppl. material 2: Fig. S1) and morphological (Piwowarczyk et al. 2017a) analyses have shown. On the ITS tree O. cicerbitae belong to clade O. subsect. Curvatae (Beck) Piwow. et al., particularly with species of the O. series Krylowianae clade (Piwowarczyk et al. 2017a). However, trees based on trnL–trnF sequences show displacement of samples of Caucasian O. flava close to O. cicerbitae (Fig. 2, Suppl. material 3: Fig. S2). A similar phenomenon is the placement of trnL–trnF sequence (Fig. 2, Suppl. material 3: Fig. S2) of O. lycoctoni near O. lucorum, a species relatively distant to Krylowianae species. This phenomenon may be explained by hybridisation and requires further research. In this case O. cicerbitae might be a species formed by the crossing of O. flava (or related species) as a female parent and one of the species belonging to ser. Krylowianae, but this supposition requires further study. In the Caucasus, both species often occupy the same habitats (humid tall herb vegetation) and grow with their hosts (Caucasalia B. Nord, Senecio L., Pojarkovia Askerova vs. Petasites Mill.) next to each other, thus facilitating gene flow.

We confirm that the newly described O. javakhetica (Piwowarczyk et al. 2018b) is distinct from the other studied species. ITS sequence does not resolve its position within other Orobanche species. However, trees based on trnL–trnF sequences (Fig. 2, Suppl. material 3: Fig. S2) show a common clade with O. gracilis (O. subsect. Cruentae Teryokhin) (BS = 98, PP = 1.00). Morphologically, there seems to be some similarity to the O. subsect. Orobanche (subsect. Galeatae sensu Teryokhin) and O. subsect. Curvatae (particularly with species of the O. ser. Krylowianae) (Piwowarczyk et al. 2018b). Finding other new species related to O. javakhetica will be helpful in confirming the phylogenetic relationships of this species.

Orobanche schelkovnikovii was incorrectly included in the O. trib./Grex Galeatae sensu Beck by Novopokrovskij and Tzvelev (1958). This erroneous determination of herbarium specimens collected by Schelkovnikov as O. caryophyllacea by Grossheim is surely the reason for its inclusion in this group. The corolla is not helmet-shaped at the apex, which clearly indicates that it should be in O. subsect. Curvatae (Piwowarczyk et al. 2019). The ITS trees indicate that O. schelkovnikovii belong to O. subsect. Curvatae and formed a clade with O. centaurina (syn. O. kochii F.W. Schultz, Zázvorka et al. 2019), O. sintenisii Beck and O. kurdica Boiss. & Hausskn. (syn. O. rosea Tzvel., Piwowarczyk et al. 2019) (Fig. 1) (BS = 99, PP = 0.98). O. kurdica is also morphologically most similar to O. centaurina, but clearly distinct based on some features and host affinity (Piwowarczyk et al. 2019). However, the sample named as O. sintenisii from Turkey (AY209276) was probably identified incorrectly. Based on host and locality it most likely belongs to O. kurdica (Fig. 1, Suppl. material 2: Fig. S1, Suppl. material 1: Table S1).

According to Novopokrovskij and Tzvelev (1958) O. grossheimii belongs to the group subsect. Curvatae, but the presence of dark coloured reddish or violet glandular hairs and morphology of the flowers may indicate that it belongs to the O. subsect. Glandulosae (Beck) Teryokhin (O. subsect. Glandulosae Novopokr., Piwowarczyk et al. 2019), which seems to be confirmed in our phylogenetic study (Fig. 1, Suppl. material 2: Fig. S1).

The newly described species O. zajaciorum (Piwowarczyk 2015) is clearly separated from other species based on ITS and trnL–trnF data, but its precise relationship to other Orobanche remains to be unclear. On the ITS tree it formed a separated clade with O. lutea Baumg. (subsect. OrobancheO. subsect. Galeatae sensu Teryokhin), however with low support (BS < 75) (Fig. 1). Morphologically, O. zajaciorum is a close relative to O. subsect. Orobanche (O. subsect. Galeatae sensu Teryokhin) – especially due to the helmet-shaped upper lip and the relatively narrowly tubular flower, however the species of O. Grex Galeatae sensu Beck (1930) are – with the exception of O. clausonis Pomel – much taller and have larger flowers, usually broad bidentate calyx segments, and different hosts (Piwowarczyk 2015).

Orobanche rapum-genistae Thuill., O. rigens Loisel. vs O. colorata/O. anatolica placed by Beck (1930) in Grex Arcuatae (O. subsect. Arcuatae Teryokhin) in our phylogenetic trees show significant discrepancy (Figs 1, 2, Suppl. material 2: Fig. S1, Suppl. material 3: Fig. S2).

Orobanche gamosepala is genetically very distinct, yet nested within Orobanche, forming a clade with O. anatolica/O. colorata (O. subsect. Arcuatae) (ITS: BS = 99, PP = 1.00, trnL–trnF: BS = 100, PP = 1.00) that is sister to the clade containing all other Orobanche species (Figs 1, 2, Suppl. material 2: Fig. S1, Suppl. material 3: Fig. S2). This species was described previously as the monotypic genus Necranthus Gilli from northeastern Turkey based on a calyx anatomy similar to Boschniakia C.A. Meyer ex Bong. and Xylanche Beck (Gilli 1968). Beck (1930) placed this species in trib./Grex Galeatae, and later Teryokhin et al. (1993) included this species in its own section O. sect. Gamosepalae Teryokh. according to its distinct calyx anatomy and seed micromorphology. In a more recent micromorphological study of Caucasian Orobanchaceae seeds, the position of O. gamosepala on the dendrogram is closer to O. colorata than to the remaining Orobanche, which is in accordance with the above results, and based on the shape of the seed cells, to Cistanche (Piwowarczyk et al. 2020b). O. gamosepala together with O. colorata and O. anatolica is one of the oldest lineages of Orobancheae (Table 2, see below).

According to some authors (e.g., Novopokrovskij and Tzvelev 1958; Domina and Raab-Straube 2010) O. colorata only grows in the countries of the Caucasus area (with ± glabrescente inflorescence), and is replaced in Turkey, Iran and Iraq by the O. anatolica s. str. (with ± lanate inflorescence). However, taxonomic and chorological restrictions corresponding to each taxon are not entirely clear (Piwowarczyk et al. 2019). In the Caucasus and Turkey (O. anatolica var. glabrescens Post and O. anatolica var. leucopogon (Boiss. & Hausskn. ex Boiss.) Beck) both individuals with glabrescente and lanate inflorescences are often found, and lanate indumenum is especially present when the plants are young, but not only. In the ITS trees (Fig. 1, Suppl. material 2: Fig. S1) O. anatolica (from Turkey) and O. colorata (Caucasus) formed a common clade but the difference between them remains unclear (BS = 100, PP = 1.00).

Orobanche raddeana is a Caucasian endemic parasitising on Campanulaceae (Campanula L., Asyneuma Griseb. & Schenk). The ITS tree may suggest that it is related to species from the subsect. Glandulosae (Fig. 1, Suppl. material 2: Fig. S1), which is also supported by morphological features, while based on trnL–trnF sequences (Fig. 2, Suppl. material 3: Fig. S2) O. raddeana forms a common clade with species mostly from the O. subsect. Curvatae ser. Krylowianae. It is worth noting that O. raddeana, described later as O. alba var. raddeana (Beck) Beck, is relatively distant to O. alba. Recently, the name O. raddeana was changed as a new species, O. campanulae Rätzel et al. (Rätzel et al. 2018), but in our opinion – despite the correction of their studies on this taxon – the traditional interpretation of the name of O. raddeana is valid and must be retained in order to avoid disadvantageous nomenclatural changes entailed by the strict application of the rules (Turland et al. 2018: Art. 14). For this reason, the authors of the new species (O. campanulae) should propose the conservation of the name O. raddeana according to ICN, even though other names, e.g., O. glabrata C.A. Mey could have priority.

Within the O. subsect. Inflatae Beck, O. grenieri (parasitic on mainly Lactuca L.) is clearly distinguished morphologically and phylogenetically from related species (O. cernua and O. cumana), as has already been shown (Piwowarczyk et al. 2015). The taxonomic relationships of the polymorphous species O. cernua and O. cumana are not entirely clear. Some researchers recognised O. cumana as a separate species, and others as varieties or subspecies of O. cernua. O. cumana parasitises cultivated plants, mainly Helianthus L. and Solanaceae (Lycopersicon Mill. L., Nicotiana L.). The problem with identifications arises when O. cumana parasitises wild species, i.e., Artemisia L. (sometimes Xanthium L.), like the closely allied typical O. cernua, because morphological differences between these two species cannot always be easily seen.

Species from subsect. Minores Teryokhin and subsect. Speciosae Teryokhin are highly polymorphic, especially regarding colour, inflorescence length and variability of flower, as well as range of hosts. In our research we used Caucasian samples of O. laxissima (a parasite of various tree species, i.e., Fraxinus L., Carpinus L., Punica L., Robinia L.), O. owerinii (a parasite of herbaceous hosts, i.e., Trifolium L., Vicia L.), and O. minor (samples from Chondrilla L. and Lactuca hosts) (Figs 1, 2). Tzvelev considered that O. owerinii is a polymorphic species, represented by several races, which can be treated as distinct species. O. owerinii is probably closely related to O. crenata Forssk. and replaces it in the upper montane zone of Asia Minor and Caucasian territories (Novopokrovskij and Tzvelev 1958). However, the typical O. crenata has not been confirmed in the Caucasus. O. laxissima seems to be very similar morphologically to O. transcaucasica Tzvel., apparently also a parasite on shrubs and trees and described in a hornbeam-oak forest from the Shemackha region in Azerbaijan by Tzvelev (1957). Rätzel and Uhlich (2004) mistakenly assigned O. laxissima to the O. subsect. Galeatae sensu Teryokhin (O. trib. Galeatae sensu Beck). Morphologically (Piwowarczyk et al. 2019) and molecularly (Figs 1, 2, Suppl. material 2: Fig. S1, Suppl. material 3: Fig. S2), the species clearly belongs to the O. subsect. Speciosae (O. trib./Grex Speciosae sensu Beck). O. laxissima can be confused by an inexperienced researcher with O. owerinii, especially in the herbarium materials without details about the host. Our results support this affinity (Figs 1, 2, Suppl. material 2: Fig. S1, Suppl. material 3: Fig. S2). Moreover, O. laxissima, O. owerinii seem to be phylogenetically similar, especially on ITS trees, with several other species. The recent diversification of these lineages (Tables 1, 2, Fig. 3) could explain why some of these species have not entirely diverged.

Molecular studies do not indicate the validity of dividing species into subsect. Speciosae because the species included here are both very morphologically and genetically similar to the subsect. Minores. Similar conclusions can be used to merit the inclusion of Vitellinae Teryokhin, Hederae Teryokhin, and Camptolepides Teryokhin in separate subsections when they are clearly similar to species from the subsect. Minores and Inflatae (respectively). The results presented here suggest that the currently distinguished systematic division of Orobanche-based morphology is frequently inconsistent with the phylogenetic studies and thus needs revision, regarding both phenotypic traits and molecular analyses, for example, the heterogeneous subsect. Curvatae is clearly resolved as polyphyletic (Fig. 1). Furthermore, our knowledge of some poorly understood species in sections in the Phelipanche genus requires further taxonomic, field (especially in Western and Central Asia), and molecular research.

Biogeography

We found strong support for Western Asia as the centre of origin for large subclades of Phelipanche, Orobanche, and Cistanche (Table 1, Fig. 3), followed by both diversification in situ as well as dispersal out of this region over the last 1–2 million years (Table 2, Fig. 3). This supports previous hypotheses that Western Asia, especially the Caucasus region and nearby high mountains in the Middle East and Western Asia, as important centres of origin for Eurasian Orobancheae (Rätzel and Uhlich 2004), despite our uncertainty in the biogeography of the ultimate common ancestor of Orobanche + Phelipanche + Aphyllon. About 30 species of holoparasitic Orobancheae are known as endemic (15 confirmed species) to the Caucasus or have most of their range there. High-mountain genetic lineages with subalpine habitats are especially unique, such as species from the ser. Krylowianae (O. inulae, O. mlokosiewiczii, O. cicerbitae, and O. arpica), as well as O. gamosepala, O. grossheimii, O. raddeana, O. javakhetica, O. schelkovnikovii, and O. zajaciorum. The extant diversity in this region is a combination of clades of recently diverged (i.e., neoendemic) species such as those in ser. Krylowianae, as well as species on relatively long branches without close relatives (e.g., O. raddeana, O. javakhetica and Phelipanche bungeana (Beck) Soják) or species-poor clades subtended by long branches (e.g., Phelypaea, or O. anatolica + O. colorata; Table 2). Thus, the Caucasus region may be considered as one of mixed endemism for parasitic Orobancheae (sensu Mishler et al. 2014). However, we do not have the temporal resolution to determine if these long-branch parasite species have always been range-limited, or have gone through expansions and subsequent contraction due to climatic or other ecological shifts.

The broader floristic and geological history of the Caucasus and high mountain region does provide some clues to the processes that its status as a centre of extant diversity, a centre of origin for large portions of this diversity, and potentially a region of mixed endemism for holoparasitic Orobancheae. The Caucasus has an unusually high proportion of endemic and relict species for a continental, non-tropical region (Tarkhnishvili 2014). Approximately 25% of vascular plant species found in the Caucasus are endemic, as well as unique vegetation types such as Colchic and Hyrcanian forests with relict tree species (Kikvidze and Oshawa 2001). The Caucasian oreoxerophytic flora has a historical connection with the Mediterranean and Asia Minor due to Pleistocene migration from Asia Minor eastwards. Following the retreat of the glaciers, xerophytic flora from the Irano-Turanian region and mountains of Central Asia also migrated to the Caucasus, with simultaneous degradation of the mesothermophilous forest vegetation (Nakhutsrishvili and Abdaladze 2005). The southern part of the Caucasus in Armenia is also located in the Irano-Anatolian biodiversity hotspot (as well as northeastern Iran and Iraq, and central and eastern Turkey). This is the only global biodiversity hotspot entirely inside Southwest Asia (Noroozi et al. 2018), with over 40% endemic plant species (Mittermeier et al. 2005). Longstanding explanations for the unique flora in this region highlight the role the Caucasus and high mountains have played as a refugium for many elements of the pre-glacial Tertiaty flora during cooling of the Pliocene and Pleistocene, and aridification during the Upper Pleistocene and Holocene (Kuznetsov 1909; Fedorov 1952; Kharadze 1960). Along with aridification, another important contributor to the flora was the Pleistocene migration of plants from Asia Minor and post-glacial xerophytic migrants from the mountains of central Asia (Nakhutsrishvili and Abdaladze 2005; Zernov 2006). Characteristics such as a dissected, heterogeneous topography, a large altitudinal range, and a relatively mild climate subsequently helped preserve these floristic elements (Kikvidze and Oshawa 2001). Consistent with this pattern, some of the oldest lineages of Orobancheae that include extant Caucasus endemics were probably also found in Western Asia at their time of divergence during the Pliocene and Pleistocene (Tables 1, 2, Fig. 3). These refugia may also explain the disjunct ranges of many Orobanche and Phelipanche species present both in Western Asia (especially the Caucasus) and further west in Europe, such as O. grenieri, P. cernua, P. portoilicitana and the Carpathian mountain species O. flava (Piwowarczyk et al. 2019).

However, we also found a number of very recent diversification events in Orobancheae, pointing to recent in situ speciation as a complementary mechanism that explains the high levels of endemism in this region (Table 2, Fig. 3). For example, the diversification of the Orobanche laxissima + O. owerinii + O. transcaucasica clade was exceptionally recent (most likely in the last 150,000 years, but this may exceed the precision of our analysis). Many species of Orobanchaceae are associated with calcareous habitats that probably favour speciation and are abundant throughout the Caucasus (Kikvidze and Oshawa 2001). Many of the same geographical and ecological factors that have made western Asia a refugium for Orobanche and Phelipanche species also likely contributed to subsequent diversification, such as topographic and habitat heterogeneity, and a diverse flora of potential host species, many of which are also endemic or of limited range. In the postglacial, continental climate of western Asia, suitable xerophytic habitat has replaced forest in many areas. The expansion of steppe, subalpine, subalpine or steppe communities – and more importantly the potential host plants occupying them – may explain the diversification of Orobancheae in this region.

By contrast, the biodiversity of Orobanche and Cistanche that evolved in Europe and especially in the Mediterranean Basin appears to have done so more recently than that in West Asia, although we cannot confidently infer ancestral states of lineages greater than 5 million years (Table 1, Fig. 3). Iberia, Italy, and the Balkans are three well-studied refugial regions of Mediterranean Europe where thermophilic species persisted through glacial periods, (Bennett et al. 1991; Comes and Kadereit 1998, 2003; Taberlet et al. 1998; Hewitt 1999; Habel et al. 2014) however our analysis is not fine-grained enough to distinguish among them. Collectively, the Mediterranean region has been recognised as another of the world hotspots of biodiversity, with more than 25,000 known vascular plant species, mostly endemic. The Mediterranean flora consists of low species-genus ratios, with many primarily long‐lived taxa restricted to island or mountain habitats, probably as paleoendemics of likely Tertiary origin (Blondel and Aronson 1999). This unique plant diversity and endemism are the result of several key factors: paleogeological and climatic history, biogeography, and ecogeographical heterogeneity, with evidence that adaptive radiation has taken place relatively recently (Blondel and Aronson 1999; Comes 2004). Several radiations within Orobanche and Cistanche have taken place over the last million years (Table 1, Fig. 3), but the clade of O. foetida Poir., O. densiflora Salzm. ex Bertol., O. sanguinea C. Presl, O. austrohispanica M.J.Y. Foley, and the more widespread O. gracilis likely began diversifying earlier, about 2–3 million years ago. Like the origin of many other Mediterranean flora elements, the ancestors of each of these clades came from western Asia, then moved into the Mediterranean and diversified (Fig. 3; Blondel and Aronson 1999). An alternative hypothesis, more strongly supported for Phelipanche than Orobanche, is that a widespread ancestor growing across Europe and western Asia may have given rise to both Western Asian and European/Mediterranean clades (the second most probable ancestral states of many Phelipanche and Orobanche common ancestors, Suppl. material 4: data S1 and Suppl. material 5: data S2).

We conclude with a cautionary note that we were not able to exhaustively sample the Orobancheae, in particular certain species of Orobanche, such as O. sect. Kotschyinae Teryokhin from the Middle East and western and central Asia. The addition of certain other lineages, such as species in O. subsect. Coerulescentes Teryokhin would likely strengthen the importance of diversification in East Asia. Finally, our results within Cistanche are sensitive to changing taxonomic concepts.

Acknowledgements

The authors thank the curators of herbaria and other people who kindly made their data and samples of the species available. Will Freyman provided computational resources.

This work was partially financed by the National Geographic grant GEFNE 192-16 (2017), Polish State Committee for Scientific Research (KBN grant no. NN303357733 (2008–2009), NN303551939 (2010–2013), the Research Projects of the Jan Kochanowski University in Kielce 612419 (2014–2017) and SMGR.20.208-615 (2020) for Renata Piwowarczyk, and statutory research funds (K/DSC/002930) of the Institute of Botany, Faculty of Biology, Jagiellonian University, Kraków, Poland.

The DNA sequence data generated and analysed during this work are available in the GenBank repository [https://www.ncbi.nlm.nih.gov/genbank/]. Alignments for the biogeography data generated or analysed during this study are available in Suppl. material 4.

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