Notes on the systematics of Cuscutasect.Subulatae (subg. Grammica) with the description of Cuscutamantiqueirana, a new species from Brazil

Abstract Cuscutamantiqueirana Costea, S.S. Silva & Sim.-Bianch. a new species from montane cloud forests of the Serra da Mantiqueira, Brazil, is described and illustrated. The morphological and phylogenetic analyses revealed that the new species belongs to sect. Subulataeofsubg.Grammica. The new species is related to C.odoratavar.botryoides, C.rotundiflora and C.globiflora from which it differs in narrower calyx lobes and the presence of four stomatiferous lobes or projections at the distal part of the ovary. A detailed morphological comparison with C.odoratavar.botryoides, morphologically the most similar taxon, is provided along with the geographical distribution, ecology and host range of the species. The morphological and phylogenetic relationships of the new species, as well as the diversity of stomatiferous projections, are discussed in the broader context of sect. Subulatae and subg. Grammica. Cuscutabolivianavar.paranensis is considered a synonym of C.odoratavar.botryoides.


Introduction
With ca. 30 species, Cuscuta sect. Subulatae (Engelm.) Costea & Stefanović is the largest infrageneric group of subg. Grammica (Lour.) Peter, and of Cuscuta L. in general (Costea et al. 2015). The section was recently circumscribed (Costea et al. 2015) based on a core of species included by Engelmann (1859) in subsect. Subulatae. This infrageneric clade has diversified in South America (Yuncker 1922(Yuncker , 1932Stefanović et al. 2007;García et al. 2014), but it also contains two African species, C. kilimanjari Oliv. (distributed across Tropical East Africa, Central Africa and Madagascar) and C. blepharolepis Welw. ex Hiern. (a more enigmatic taxon known only from two collections in Guinea and Angola, in Western Africa). Although C. blepharolepis has not yet been sampled, C. kilimanjari was found to be nested deeply within this clade, as sister to C. cristata Engelm. (S. Brazil to N. Argentina), strongly suggesting long-distance dispersal (Stefanović et al. 2007;García et al. 2014). Flowers of sect. Subulatae are among the largest in Cuscuta, often fleshy, and in many species apparently cross-pollinated (Wright et al. 2012). Infrastaminal scales, which are unique structures with defence role in Cuscuta flowers (Riviere et al 2013), are very diverse in shape, size and number of fimbriae; in a few species they are entirely reduced while in others they possess densely papillate fimbriae (Riviere et al. 2013). Pollen is also more varied among species than in other sections of subg. Grammica; it can be 3, 4, 5 or 6-7-colpate, and tectum can be imperforate, perforate, microreticulate, or reticulate (Welsh et al. 2010). Styles are thick, cylindrical or subulate, and stigmas are large, convoluted and lobed (Yuncker 1932;Wright et al. 2011). The fruit is usually dehiscent (Ho and Costea 2018) with large seeds (Olszewski et al. 2020). Plastome evolution studies have revealed extensive losses of plastid genes, including the otherwise highly conserved small and large ribosomal subunits (Braukmann et al. 2013). Engelmann (1859), Yuncker (1921Yuncker ( , 1922Yuncker ( , 1923Yuncker ( , 1932 and Hunziker (1947Hunziker ( , 1949Hunziker ( , 1950 described the majority of taxa in sect. Subulatae, but this clade has not been revised at species level to date. Among the practical obstacles towards a taxonomic revision of this section are the scarcity of herbarium material available and notoriously difficult DNA extraction from herbarium specimens. After plants dry, they often become brown-blackish and more difficult to analyze morphologically. Plastid sequences, which have been used extensively to reconstruct phylogeny of the entire genus , subgenus Grammica (Stefanović et al. 2007), and multiple clades of the latter subgenus (e.g., Costea et al. 2008Costea et al. , 2011aCostea et al. , 2011bCostea et al. , 2013Costea et al. , 2020Costea and Stefanović 2009), cannot be employed for this section because of the plastome reductions (Braukmann et al. 2013).
The objective of this study is to report a new species in sect. Subulatae, as well as to discuss its putative relationships with other taxa in this group. The new species has been discovered independently both among herbarium specimens and by doing field work.

Molecular phylogenetic analyses
Of the 28 collections of individuals belonging to the new species C. mantiqueirana, three specimens (Appendix 1) were found to be of sufficient quality and quantity for molecular studies. To infer the phylogenetic affinities of this species within Cuscuta sect. Subulatae, we obtained sequences from the internal transcribed spacer (ITS) region of nuclear ribosomal DNA (rDNA). DNA extractions, polymerase chain reaction (PCR) reagents and conditions, amplicon purifications, cloning, and sequencing procedures follow Stefanović et al. (2007) and Stefanović and Costea (2008). The sequences generated in this study have been submitted to GenBank (accession numbers MZ389688-MZ389691). Using Se-Al v.2.0a11 (Rambaut 2002), newly obtained sequences were incorporated into previously aligned nrITS matrix of accessions from Cuscuta sect. Subulatae (Stefanović et al. 2007;Stefanović and Costea 2008;deposited in TreeBASE under study number S1929). Based on these, more inclusive analyses, we selected C. microstyla Engelm. as a functional outgroup.
Phylogenetic analyses were conducted under parsimony and maximum likelihood using PAUP* v4.0b10 (Swofford 2002). Sequence data were treated as unordered and all changes were equally weighted. Gaps in the alignments were treated as missing data. Given the moderate number of terminal units, the parsimony searches were conducted with a Branch-and-Bound algorithm, ensuring recovery of all of the most parsimonious (MP) trees. The full heuristic searches for maximum likelihood (ML) trees were performed under the general time-reversible (GTR) model of DNA substitution (Lanave et al. 1984), with the rate of variation among nucleotides following a discrete gamma distribution and allowing for invariable sites (GTR+G+I), involving 100 replicates with stepwise random taxon addition, tree bisection-reconnection (TBR) branch swapping, and MULTREES option on. All model parameters were estimated from data, except the base composition, where empirical frequencies have been used. The support for clades was inferred by nonparametric bootstrapping (Felsenstein 1985), under parsimony, using 1,000 heuristic bootstrap pseudoreplicates, TBR branch swapping, and MULTREES option on. Support for a relationship was considered weak if bootstrap value was < 70%, moderate if between 70 and 90%, and strong if > 90%.

Microscopy
Flowers, fruits and seeds removed from herbarium specimens were steeped in gradually warmed 50% ethanol, which was then allowed to boil for a few seconds to rehydrate tissues. An ethanol solution is more suitable for rehydration than simple water because it removes some of the dark pigments that result after drying, and at the same time, it hardens the tissues, which are very delicate in the Cuscuta flowers. For basic morphology, flowers were dissected under a Nikon SMZ 1500 stereomicroscope and imaged with PaxCam Arc digital camera equipped with a PAX-IT 8.2 (MIS Inc. 2021, Villa Park, Illinois) imaging software. Numerous photographs illustrating details of the floral and fruit morphology for all taxa, including their type collections, are made available on the Digital Atlas of Cuscuta website (Costea 2007-onwards). To examine finer (micro)morphological features, rehydrated flowers, fruits and seed samples were dehydrated through an ethanol series (50%, 70%, 85%, 95%, and 100%; each step 1h) and then critically point dried with Tousimis Autosamdri-931. Samples were mounted on aluminum stubs and sputter-coated with 30 nm of gold using Emitech K550 sputter coater. Examination, imaging and measurements were made using a Hitachi SU1510 scanning electron microscope (SEM) at 5-10 kV.

Molecular phylogeny
The parsimony analysis resulted in 12 MP trees [length = 161; consistency index (CI) = 0.907; retention index (RI) = 0.957]. The maximum likelihood analysis resulted in a single ML tree, topologically fully compatible with the strict consensus of the MP trees. The ML phylogeny was selected to illustrate the inferred relationships in this section, including the placement of C. mantiqueirana, as well as branch lengths (Fig. 1). Based on the strong support, as measured by bootstrap values, and the sequence divergence, as indicated by the branch lengths, molecular data revealed three major lineages within Cuscuta sect. Subulatae (Fig. 1). These results are fully consistent with our previous findings (Stefanović et al. 2007;Stefanović and Costea 2008). Cuscuta microstyla formed a distinct lineage within sect. Subulatae (Fig. 1). This Chilean-Argentinean species is restricted to high Andes, and represents an exception in the section in its relatively small flowers and indehiscent fruits. The second lineage (Clade A; 95% BS; Fig. 1) consisted of species (e.g., C. chilensis Ker Gawl., C. cockerellii Yunck., C. foetida Kunth, C. odorata Ruiz & Pav. var. odorata, C. paitana Yunck., C. purpurata Yunck.; Fig. 1) with tubular-campanulate flowers, papillate infrastaminal scales (Riviere et al. 2013), and 3-colpate pollen grains (Welsh et al. 2010) primarily distributed along the Andes (Ecuador, Peru, Bolivia, Chile). Finally, the third lineage (clade B; 100% BS; Fig. 1) that also included C. mantiqueirana, is characterized by flowers with rotate or globose to urceolate corollas, infrastaminal scales without or with only a few papillae (Riviere et al. 2013), and usually (although some exceptions are known) 4-6-colpate pollen grains (Welsh et al. 2010). This lineage includes C. cristata Engelm., C. globiflora Engelm., C. friesii Yunck., C. grandiflora Kunth, C. kilimanjari, C. parodiana Yunck. ( Fig. 1; clade B), and most likely, based on their morphological similarity, several other species that could not be included in the molecular analysis: C. odorata Ruiz & Pav. var. botryoides Engelm., C. rotundiflora Hunz., and C. boliviana Yunck. Members of this morphologically identified clade occur east of the Andes in Argentina, Uruguay and Brazil, but a few are found along the Andes (Colombia to Chile). An identification key for the taxa of clade B is included below. Among these taxa, C. mantiqueirana is morphologically most similar, and geographically closest to C. odorata var. botryoides, and a comparison between the two taxa is provided in Table  1. Based on our current sampling, C. mantiqueirana is reciprocally monophyletic and molecularly distinct from other members of this clade, as evidenced by the branch length subtending it and strong bootstrap support (Fig. 1). The molecular results agree with the morphological distinctiveness of all the species considered, suggesting that taxa for which DNA could not be extracted, but which are morphologically distinct, will also be validated as discrete lineages when molecular data become available.
Geographical distribution and ecology. The new species is apparently endemic to Serra da Mantiqueira in Southeastern Brazil (states of Minas Gerais, Rio de Janeiro and São Paulo), where it occurs at elevations between 800-2360 m. The climate is mesothermic, characterized by distinct dry and rainy seasons, with an average temperature (4-) 5-7 mm 3.8-5 mm Calyx 2.8-4 mm long, divided 2/3-4/5 to the base, tube 0.5-1.5 mm long, lobes 2-2.5 mm long, ovate-oblong, longer than wide to as long as wide, the two external ones overlapping, usually not carinate, not auriculate at base.

Ovary
Ovary apex on both sides of styles risen to form 4 lobes or projections with stomata, 0.4-0.6 mm long.
Apex without lobes or projections but a few stomata may be present.

Styles and stigmas
Styles 0.3-0.9 mm long, 0.25-0.5 mm thick, cylindrical or slightly subulate, sometimes also with stomata at their base; stigmas 0.35-0.5 mm long and 0.5-0.7 mm wide  Ururahy et al. 1983;Veloso et al. 2012). During field work conducted in Itatiaia National Park and Monte Verde, we observed C. mantiqueirana at forest edges, margins of roads and clearings as well as parasitizing isolated woody plants in the forest (Fig.  4A, B). The most common host is Fuchsia regia (Vell.) Munz (Onagraceae; Fig. 4E), which is the most widely distributed species of this genus in Brazil, occurring throughout the distribution range of C. mantiqueirana (Berry 1989). Fuchsia regia is currently accepted to include three subspecies, regia, serrae P. Berry, and reitzii P. Berry (Berry 1989), and future field work will have to determine the frequency of occurrence on these subspecies. To a less extent, perhaps as secondary hosts, C. mantiqueirana parasitizes other herbaceous or woody plants: Asteraceae (Baccharis L., Lepidaploa (Cass.) Cass., Mikania Willd., including Mikania micrantha Kunth, and other unidentified  Phenology. Flowering in Nov-Dec and Feb-Aug, which may depend on the elevation. Very few herbarium specimens possess capsules and seeds which suggests that plants are preponderantly xenogamous and also reproducing vegetatively (Wright et al. 2012).
Etymology. The specific epithet is a feminine adjective that comes from the name of the mountain range to which the species is apparently endemic. The word "Mantiqueira" is derived from Tupi-Guarani meaning "mountains that cry" alluding perhaps to the plethora of dripping water, streams and rivers that are present during the wet season with abundant rainfall (Mendes Júnior et al. 1991).
Vernacular names. The common names used in the area are: Cipó-chumbo, fiosde-ovos, erva-de-passarinho (although also commonly used for other species of Cuscuta that occur in the region).
Provisional conservation status. The GeoCAT rapid assessment tool (Bachman et al. 2011)   The morphological distinctiveness of C. mantiqueirana allows its unequivocal recognition as a new species even though we could not obtain molecular data for some morphologically similar taxa -C. rotundiflora and C. odorata var. botryoides. The available molecular results agree with the morphological patterns observed in section Subulatae, and the similarity of C. mantiqueirana, C. odorata var. botryoides, and C. rotundiflora strongly suggests a phylogenetic proximity of these taxa in clade B (Fig. 1). Nevertheless, a complete picture of the evolutionary relationships and systematics of this clade will require molecular results for all the taxa involved.
As indicated, C. mantiqueirana is most similar morphologically to C. odorata var. botryoides (Table 1), a taxon that was described by Engelmann (1859) from "Southern Brazil" based on a single specimen, Lobb 49 (K, MO). Engelmann (1859) viewed this variety as "intermediate" between C. odorata and C. chilensis. Subsequently, Yuncker (1922Yuncker ( , 1932 maintained this taxon as a variety of C. odorata, but compared it with C. globiflora. More recently, Hunziker (1947) described C. boliviana var. paranensis Hunz. from Misiones (Argentina) and Paraná (southern Brazil), which he considered to be most similar to C. boliviana (var. boliviana) and C. cristata. These two varietal names have been accepted by modern floristic overviews (e.g., Zuloaga et al. 2008; Flora do Brasil 2020), but the taxa themselves have remained little known until now. After examining the types and few specimens available for C. odorata var. botryoides and C. boliviana var. paranensis, we consider these names synonymous. Variety botryoides has priority at this rank ("the original subdivisional epithet", a rule that had already been in place in 1947 when it was described by Hunziker (Art 55; Camp et al. 1948). Although it could not be included in the molecular study, C. odorata var. botryoides is likely not related to C. odorata var. odorata because at least some of its pollen grains are 4-5-colpate and its infrastaminal scale fimbriae have only 1-2 distal papillae. Thus, C. odorata is most probably polyphyletic; var. odorata shares affinities with the species of clade A (comprised of C. foetida, C. purpurata, C. chilensis, etc., Fig. 1), while var. botryoides is a member of clade B. The taxonomic rank and evolutionary relationships of var. botryoides remain to be solved by a future study.

Stomatiferous protuberances
Stomatiferous structures have been recently documented in many Cuscuta species (reviewed by Clayson et al. 2014); however, their presence at the apex of the ovary and base of styles in C. mantiqueirana is a novel feature. Stomatiferous (multicellular) protuberances (SPs) develop during anthesis on the haustorial stems of species in subgenus Grammica, as well as on the calyx and corolla of flowers in species from multiple clades of subgenera Cuscuta and Grammica (Clayson et al. 2014). When present on the calyx and corolla, SPs are diversely shaped (e.g., tubular, hemispherical, conical, crests) and have evolutionary and taxonomic significance Costea et al. 2011aCostea et al. , 2011bCostea et al. , 2013. SPs on the flowers are characteristic of species that have evolved in areas with a marked dry season (Clayson et al. 2014). The water loss through the SPs stimulates the hosts to absorb more water by increasing the negative pressure/tension in the xylem of the host, via the haustoria connection (Clayson et al. 2014). Some taxa of section Subulatae also have SPs on the calyx lobes; for example, the species epithets of C. cristata and C. alatoloba Yunck. refer to the presence of crests on the calyx lobes, but their authors (Engelmann 1859;Yuncker 1932) did not know that these structures bear stomata or their function. The discovery of stomatiferous structures on the ovary and style base of C. mantiqueirana improves the knowledge about the diversity of SPs, and strongly suggests that the morphology of floral SPs will play a significant role in the species-level systematics of section Subulatae.