Two new Malagasy species of genus Piper L. (Piperaceae), Piper malgassicum and Piper tsarasotrae , and their phylogenetic position

: Here we describe two new species of genus Piper L. from Madagascar: Piper malgassicum and Piper tsarasotrae , the species names referring to the currently known distribution areas. Th ese two species contribute, at least in part, to the production of local voatsiperifery pepper, probably mixed together with P. borbonense , and are therefore economically relevant as spices. We used a selected set of characters (those more easily observable on herbarium samples) for principal component analysis to assess the relative distance between the species, including the analysis of the autochthonous species of Piper known from Africa and Madagascar. In order to check the identity and assess the phylogenetic position of the two species, we also sequenced the chloroplast gene ndh F, the trn L intron, and the nuclear gene G3pdh . On the basis of these results we show here the relationships between these two new Piper taxa and the most closely related species within the genus (excluding P. heimii and P. pachyphyllum , for which only morphological characters were available).

Only two endemic species are currently known for the African continent: P. guineense and P. capense. Piper guineense is a dioecious vine, relatively similar to the majority of southwestern Asian species, whereas P. capense is a shrub with bisexual flowers, hence resembling many species of the American continents . The knowledge of the genus in Madagascar is far from complete. Currently, P. heimii C.DC. and P. pachyphyllum Baker are indicated for the island, while P. borbonense (Miq.) C.DC. was described for the island called at that time Île Bourbon, currently La Reunion (Weil et al., 2017), belonging to the Mascarene Islands, 600 km east of Madagascar. Its presence in Madagascar is a matter of debate, even if De Candolle (1923,1869) had assigned some samples from Madagascar and Mauritius to this species (see Appendix 1 about herbarium samples from the site http://www.caryologia.unifi.it/tjb/Appendix1. pdf). However, this species is cultivated, which makes it more difficult to assess its natural distribution.
Here we describe two new species of Piper L. from Madagascar on the basis of their morphology, supported by molecular data. Both species are mixed with P. borbonense in the so-called high-quality spice voatsiperifery pepper.

Morphological characters analysis and PCA
Herbarium samples were prepared, from which the type specimens were chosen (see Appendix 1 with herbarium samples saved at the permanent link http://www. caryologia.unifi.it/tjb/Appendix1.pdf). A number of characters were observed and measured (where necessary) with a stereomicroscope. The herbarium samples were stored by the Tropical Herbarium of Florence (FT, Centro Studi Erbario Tropicale, Università degli Studi di Firenze).
Twenty-one characters (those that showed variation) were coded in a matrix (Table 1) and used as input for the principal component analysis (PCA) with the software PAST 3.16 (Hammer et al., 2001).

Anatomical characters
Inflorescence stems were cut with a blade, stained with 1% phloroglucinol (w/v) in 12% HCl for 5 min, and observed with a bright field light microscope to stain lignin (as in Mosti et al. 2012).

DNA extraction
For the DNA extraction, leaf samples were collected from the tropical forest of Vohiday (samples PNsv1-10, Table  2) and from the Tsarasotra area (samples PNst1-10, Table  2). Plant tissue samples were conserved and transported inside 20-mL plastic tubes filled with 96% ethanol (Murray and Pitas, 1996;Bressan et al., 2014).
For DNA extraction, 40 mg of dry leaf sample was placed into a 2-mL tube with tungsten carbide beads, frozen in liquid nitrogen, and finely ground in a tissue homogenizer (Tissue Lyser, QIAGEN). DNA was extracted using an Invisorb Spin Plant Mini Kit (Stratec Molecular). Amplification of the trnL intron and the low-copy nuclear gene G3pdh followed the protocols of Taberlet et al. (1991) and Strand et al. (1997), respectively. A set of four primer pairs were designed using the chloroplast genome sequence of Piper kadsura (GenBank: KT223569.1) to cover the entire ndhF gene.
The new DNA sequences produced during our investigation were deposited in GenBank (GenBank accession numbers are indicated in Table 2).

Phylogenetic analysis
Together with the new sequences produced here, other sequences used are available in GenBank, more specifically those of genus Piper used by Smith et al. (2008). We used one species of Peperomia (Peperomia pellucida) and Houttuynia cordata as outgroups on the basis of phylogenetic analysis of Piperaceae by Jaramillo and Manos (2001) and Wanke et al. (2007) showing that Peperomia is sister group to Piper s.l., while Houttuynia is more distantly related to both of these genera (see, for instance, fig. 5 in Wanke et al., 2007). Optimal multiple alignment was obtained with CLUSTALW 1.81 (Thompson et al., 1994). The matrices for each of the three gene sequences employed were combined with Python (Python version 2.6.4; Biopython 1.57) program combinex2_0.py, written by Papini (Lewke Bandara et al., 2013;Simeone et al., 2016), released under GPL license and available at www.unifi.it/caryologia/ PapiniPrograms.html.
A maximum likelihood (Felsenstein, 1981) search was done by preliminarily using MrMODELTEST 2.0 (Nylander, 2004) to evaluate the best likelihood model on the basis of the Akaike information criterion (Akaike, 1974). The model was used as settings for Bayesian inference with the program MrBayes 3.4b4 (Huelsenbeck and Ronquist, 2001;Ronquist et al., 2012). A maximum likelihood phylogenetic analysis was done with RaxML (Stamatakis et al., 2012) and the resulting trees were edited with Figtree (Rambaut, 2009). We mapped the support on the tree branches with the results of the Bayesian phylogenetic analysis as follows: after the 'burn-in' trees were removed from the dataset as in Papini et al. (2007, Table 2. Geographical coordinates of the samples collected for DNA extraction. GenBank accession numbers of the corresponding G3pdh, trnL, and ndhF are reported on the right side of each accession. All the samples of P. tsarasotrae come from the locality Tsarasotra, Ambositra region (Madagascar), while all the samples of P. malgassicum come from the Vohiday forest, Ambositra region (Madagascar). Latitude and longitude of collection places are indicated underneath the species name.  . The remaining trees were used to produce a 50% majority-rule consensus tree with PAUP, in which the percentage support was considered equivalent to Bayesian posterior probabilities.

Morphological characters analysis and PCA
The characters used for the species description and for comparison were observed and measured with a stereomicroscope on herbarium samples of the two new species and of the most closely related species of Piper (images of the samples and original protologues can be found in Appendix 1: herbarium samples). Also in Appendix 1 (http://www.caryologia.unifi.it/tjb/ Appendix1.pdf), a list with the investigated samples (scanned samples, in the majority of cases) of other species is reported. The characters were coded as numeric states (Table 1) and analyzed with PAST. PCA was based on a set of characters, those most variable and easily observable on herbarium samples. The results of the PCA analysis are shown in Figure 1. The samples from Tsarasotra (from now on, P. tsarasotrae) were quite isolated, even if quite close to P. guineense and to the samples from Vohiday (from now on, P. malgassicum) and P. heimii (Figure 1). Figure 1 also shows that P. pachyphyllum and P. borbonense are relatively close.

Phylogenetic analysis
Phylogenetic analysis (Figure 2) showed that P. malgassicum and P. tsarasotrae are strictly related to each other and to P. borbonense, this last clustering together with P. malgassicum with 100% Bayesian support (BS).
These two species formed a monophyletic group with P. tsarasotrae with 70% BS. The sister group of this cluster was a group of 5 sequences of the African species P. guineense (85% BS), while the Asian species P. caninum formed the sister group to the former species, but with BS of less than 50% (Figure 2).

Microscopy observations
Observation with a stereomicroscope was useful for the study of the micromorphological characters of the flowers necessary for the following description. In P. tsarasotrae male flowers, usually three (very rarely four) stamens are present ( Figure 3A), with two anthers with lateral apertures ( Figure 3A1). In the female flowers, the number of stigmas may vary from 3 to 4. In Figure 3B, a case with three lobed stigmas is shown. In P. malgassicum male flowers, stamens are sometimes solitary ( Figure 3C) and show two anthers with lateral apertures ( Figure 3C1). In female flowers, stigmas are most frequently three, sometimes four, still visible on the enlarged fertilized ovary ( Figure 3D). The stigmas are sessile ( Figure 3D1).
The observation with a light microscope of crosssections of the stem showed that in P. tsarasotrae two circles of bundles are present ( Figure 4A): a group of larger, more internal bundles and an external group of smaller bundles ( Figure 4B).
Also, in P. malgassicum, the inflorescence stem in cross-section showed two circles of bundles ( Figure 4C): a group of larger, more internal bundles and an external group of smaller bundles ( Figure 4D). In this species, a continuous layer of sclerenchyma enclosed the smaller bundles ( Figure 4C).

Description of the two new species
The morphological nomenclature employed here follows that of Simpson (2010).   figure) are evidenced in green, together with the genetically strictly associated P. borbonense. The phylogenetically close P. guineense accessions are evidenced in red. All names refer to species of genus Piper L. with the exception of Peperomia pellucida and Houttuynia cordata, whose names are reported entirely (together with the genus name) with the provenance on the right.  instead of cordate. Dioecious. Shrub, sometimes epilithic and sometimes creeping on the ground, swollen stem nodes. Leaves alternate. Shape oblong ovate, 4.5-8 cm long and only 0.5-2 cm wide. Leaf apex acuminate, while the leaf base is uneven and acuminate. Inflorescence leaf opposite, cylindrical, and erect. Female spikes 4-6 cm long, with a peduncle 1-2 cm long, with small sessile spirally arranged flowers. Single ovary, 4 (rarely 3)-lobed white stigma, covered by short appendages. Male spikes 3-5 cm long, with a peduncle 1-2 cm long and stamens organized in groups of three. Ripe fruit reddish and rounded, 0.4-0.7 mm long, fruit pedicel 0.8-1.2 cm. Each fruit gives off a single rounded-shaped seed. Inflorescence stem in crosssection with two circles of bundles: a group of larger more internal bundles and an external group of smaller bundles.

Piper tsarasotrae Papini, Palchetti, Gori & Rota
Living in arid forest. The environment of the species is shown in Figures 5A and 5B, while the female cones are shown in Figures 5C and 5D. Fruits in Figure 5E. In Figure  5F, both the inflorescence and the fruits can be observed on the same individual. Usually three (very rarely four) stamens present with two anthers with lateral apertures. Number of stigmas from 3 to 4.
Similar to Piper borbonense (Miq.) C.DC. but differing since its foliar basis is uneven and roundish instead of cordate. Dioecious. Liana climbing up to 10-15 m. Leaves alternate. Shape ovate-elliptic, 6.5-8 cm long and 3-5 cm wide. Presence of adventitious roots for climbing at the nodes. Heterophylly: the lower part of the stem showing cordate leaves. Leaf apex acuminate, while the leaf base is uneven and rounded. Inflorescence leaf opposite, cylindrical and erect. Female spikes 3-8 cm long, with a peduncle 1-2 cm long, with small sessile spirally arranged flowers. Single ovary, 3-4-lobed white stigma. Male spikes 6-10 cm long, with a peduncle 2-3 cm long and stamens organized mainly in groups of two. Ripe fruit reddish and oval, 0.4 cm long, fruit pedicel 0.8-1.2 cm. Each fruit gives off a single rounded-shaped seed.
In Figure 6A the cordate leaves of the lower part of the stem are shown. Figure 6B shows the collection of the plant's climbing trees up to 10-12 m. Fruits are shown in Figures 6C and 6D. Male inflorescence is shown in Figure  6E and adventitious roots are visible in Figure 6F. Female inflorescence is shown in Figure 7A, fruits in Figure 7B, and number of stigmas in Figure 7C. Stamens sometimes solitary with two anthers with lateral apertures. Stigmas most frequently three, sometimes four, still visible on the enlarged fertilized ovary. Stigmas are sessile. Inflorescence stem in cross-section with two circles of bundles: a group of larger, more internal bundles and an external group of smaller bundles. Continuous layer of sclerenchyma enclosing the smaller bundles. Living in humid forest.

Discussion
Morphological results show that the description of P. tsarasotrae does not overlap with the description of the other species known to be indigenous to Madagascar, such as P. heimii (quite close to P. malgassicum ) and P. pachyphyllum. Piper heimii appears to be very close to P. malgassicum, but the first has lanceolate-ovate leaves 12.5 cm long according to the protologue, whereas the second has ovate leaves 6.5-8 cm long. However, P. pachyphyllum and P. heimii have not been recently sampled and should be further investigated.
Phylogenetic analysis of the two new species in the context of a subset of the matrix used by Smith et al. (2008) showed that P. malgassicum and P. tsarasotrae belong to a clade comprising P. borbonense, P. guineense, and P. caninum. The same clade was also identified by Smith et al. (2008) with higher Bayesian support with respect to our phylogenetic analysis. Possibly, the larger sampling in this group due to the insertion of P. malgassicum and P. tsarasotrae decreased the robustness (however, considerably high at 85%). The placement in a monophyletic group formed by P. malgassicum, P. tsarasotrae, and P. borbonense (relatively close to P. pachyphyllum in Figure 1) in the phylogenetic analysis is also corroborated by the biogeographical position of these entities, since the first two species are endemic to Madagascar, while P. borbonense originates from La Reunion and Mauritius (but is also present in Madagascar according to De Candolle (1923)). The possible presence of P. borbonense in Madagascar, not only in cultivated form but also as a spontaneous species, also as possible further component of voatsiperifery pepper, should be ascertained.
The two new entities appear to belong to Piper s.s. in the sense of Jaramillo et al. (2008).
Piper malgassicum is probably more closely related to P. borbonense and P. heimii than to P. tsarasotrae, even if P. malgassicum appears to be wild in Madagascar, while P. borbonense may have been introduced on this island for spice production. Piper tsarasotrae has a completely different ecological niche (creeping on the soil and on the rocks, sometimes lianous, but on low plants) with respect to P. malgassicum, which is a more typical forest lianous species of Piper. These three species appear to be phylogenetically related to P. guineense, endemic to the African mainland. This genetic affinity was already indicated by Jaramillo et al. (2008) and appears to be more strict between P. tsarasotrae and P. guineense.
The presence of two circles of vascular bundles in the stem of many Piper species was defined as polystelic organization by Isnard et al. (2012) and was considered by these authors as a synapomorphy of the family Piperaceae with the exception of the genus Verhuellia. This character was observed in detail, for instance, in some American species such as P. amalago L. (Dos Santos et al., 2015), in which a continuous layer of sclerenchyma was also described, as in P. malgassicum. This scheme is typical of the investigated species of Piper (Dos Santos et al.,

2015)
, but P. tsarasotrae shows some difference, since the sclerenchymatic layer is not continuous.
In conclusion, the two new species of Piper described here concur in the production of some of the locally produced voatsiperifery pepper, probably together with P. borbonense, and are hence of economic importance as spices. The association of species morphological identification with DNA sequences could be useful as a bar-coding method for identification of the components of spices and drugs in traditional mixtures (Chaveerach et al., 2006).