A new species of Cissampelos (Menispermaceae) from Bolivia and Paraguay

Abstract The new species Cissampelos arenicola M. Nee & R. Ortiz, from the Bolivian and Paraguayan Chaco is described, its affinities are discussed, and its preliminary conservation status is evaluated. The species is at present known from 13 collections from sand dunes or dry forests. Cissampelos arenicola is distinguished from all other American species in the genus by its ovate- to subreniform-trilobed leaves, 8-locular synandria, and relatively large, and scarcely ornamented endocarps. The most common perianth condition in the pistillate flowers of Cissampelos is one sepal and one antesepalous petal, and while these may vary in number, they are always found adaxial to the carpel, and although the southern African taxon called Cissampelos capensis, whose generic position is uncertain, superficially resembles Cissampelos arenicola, its sepals and petals are consistently lateral to the carpel and not adaxial.


Introduction
Th e pantropical genus Cissampelos L., together with African Antizoma Miers and the mostly Asian Cyclea Arn. ex Wight, were placed in subtribe Cissampelinae, one of the three subtribes in tribe Menispermeae, which together with seven other tribes were recognized by Diels (1910) in the family Menispermaceae. In studies based on ndhF, matK, trnL-F, ITS, rbcL, and atpB sequence data, subtribe Cissampelinae has been consistently recovered as monophyletic (Ortiz et al. 2007, Wang et al. 2007, Hoot et al. 2009, Jacques et al. 2011, although relationships within the subtribe remain unresolved (Jacques et al. 2011). Th e sampled species of Cissampelos form a clade that is sister to Cyclea (Ortiz et al. 2007, but studies including the southern African Cissampelos capensis L. f. recovered a polyphyletic Cissampelos, with Cyclea (Hoot et al. 2009, Jacques et al. 2011, and Antizoma (Jacques et al. 2011) nested within. While Cissampelos capensis is recognized in Cissampelos (Botha 1980), at times it has been placed in Antizoma (Diels 1910), which is characterized by a shrubby habit, a spur borne along the stem [abaxial to the petiole], pistillate fl owers with two sepals on opposite sides of the carpel, and two antesepalous petals (Miers 1851). Th e most common perianth condition in the pistillate fl owers of Cissampelos is one sepal and one antesepalous petal, occasionally, more than one sepal and petal are found in some of the species (Diels 1910). Based on the fact that some species of Cissampelos display variation in sepal and petal number, the shrubby C. capensis was removed from Antizoma and included in the genus Cissampelos (Botha 1980). However, commonly overlooked is that while the pistillate fl owers of Cissampelos may show variation in the number of sepals and petals, these are consistently located facing the adaxial slit of the carpel (Fig. 1A). On the other hand, the sepals and the antesepalous petals of Antizoma are usually located at both sides of the adaxial slit of the carpel (i.e., on the lateral sides of the carpel) (Fig. 1B), or around the carpel when more than two sepals/petals are present. Th e pistillate fl owers of C. capensis have not only two sepals and two antesepalous petals but these are located in a similar manner as in Antizoma angustifolia Miers ex Harv. and in A. miersiana Harv. At present, the taxonomic status and phylogenetic affi nities of Cissampelos capensis are unclear, similarly, the generic boundaries and relationships between Cissampelos, Antizoma, and Cyclea remain unresolved, and a re-assessment of all the genera in subtribe Cissampelinae is warranted, but is beyond the scope of this paper. Until further studies clarify the taxonomy and affi nities of Cissampelos capensis, here it is still provisionally referred to as Cissampelos and we compare it to the new species C. arenicola as they both share superfi cial morphological resemblance in habit and foliage.
Th e last comprehensive revision of the genus Cissampelos recognized 20 species, nine of them distributed in the Americas, nine in Africa, one in Asia, and the pantropical Cissampelos pareira L. (Rhodes 1975). Species of Cissampelos are often found growing in open, disturbed habitats, and with a few exceptions, are morphologically variable, such that species limits are frequently diffi cult to discern. As new collections have accumulated since Rhodes's (1975) revision, it has become evident that a number of collections from similar habitats in Bolivia and adjacent Paraguay, are conspecifi c but diff er from all other described American species of Menispermaceae. Still, placing this material generically has been challenging. Because of the general similarity in leaf shape with the North American Cocculus carolinus (L.) DC., the specimens were initially identifi ed as belonging to Cocculus DC. However, the Old World-cen- tered Cocculus (Diels 1910, Troupin 1962, has only a few species in temperate and subtropical North America and Mexico, but none in South America. Examination of the minute staminate fl owers of these Bolivian and Paraguayan plants allowed for the specimens to be confi dently placed in Cissampelos, and the new species is described here as Cissampelos arenicola.

Materials and methods
We studied all 13 herbarium collections of the new species housed at MO and at NY. Most of the collections have duplicates in several other herbaria but these were not available during this study. Th e specimens studied include male, female, and fruiting individuals, an infrequent situation in dioecious plants, such as Menispermaceae. Additionally, for comparison, we examined specimens for two other species that share some morphological similarities with the new species. Th ese included: the Paleotropical C. mucronata A. Rich. (fi ve specimens) and the southern African C. capensis (seven specimens). Measurements given in the species description refer to ranges of the mean values stemming from two to three replicate measurements of each structure and organ per individual voucher specimen. Before measuring, fl oral parts were fi rst rehydrated, and endocarps were fi rst boiled in water for a few minutes and the fl eshy part of the fruit removed. Menispermaceae fruits develop asymmetrically so that the long axis do not necessary correspond with fruit length; endocarp measurement follow the convention of Ortiz (2012).
Distribution and ecology. Th e species is at present known from southern Bolivia and northwestern Paraguay (Fig. 4). All Bolivian collections are from similar habitats of the sandy dune systems southwest of the city of Santa Cruz on the main highway which runs south to Abapó and on to Camiri and to Yacuiba on the Argentinian border. In Paraguay the species was collected along ruta Transchaco and also near the border with Bolivia in the proposed National Park Médanos del Chaco, where it has been found in seasonal forests and in dunes. In Bolivia there are extensive sandy savannas and large active dunes, the most well-known being the "Lomas de Arena" recreation area 15-20 km SSE of the center of Santa Cruz de la Sierra. Th e area where the species has been collected is on the western edge of this dune fi eld. Plants were collected from 300-470 m elevation.
Pollination. No observations of pollinators are available, nor notes on any possible odor. Th e extremely small size of the fl owers suggests that a very small insect must be involved in pollination. Label note of Nee 51401: "It is diffi cult to separate the sparse and inconspicuous fl owering and immature fruiting material from the vegetative mass".
Phenology. Male fl owers were collected in February, May, November, and in December, pistillate fl owers and mature fruits in February, April, June, and in December.
Etymology. From the Latin, arenicola, dweller in sand, as the species seems to be restricted to sandy savanna soils and edges of dunes.
Preliminary conservation status. Cissampelos arenicola is at present known from 13 collections at nine localities from southern Bolivia and northwestern Paraguay. Th e sandy savannas in the area surrounding Santa Cruz de la Sierra are frequently burnt and heavily grazed by cattle. Th is appears to have little eff ect on the native vegetation, but development of the land for subdivisions and chicken ranches is a greater threat and is destroying much of the original vegetation. Although the Viru-Viru pampa surrounding the international airport north of the city of Santa Cruz is maintained as a natural savanna, C. arenicola has never been collected there. In Paraguay, C. arenicola has been collected in habitats described as seasonal forest, but also in areas with nomadic sand dunes. Th us, the "ephemeral" condition described on the labels may refer to its dynamic and transient habitat and not to the plant per se. Because of the fragmented and threatened habitat in Bolivia, its reported ephemeral condition of its habitat in Paraguay, and its usually small population sizes, C. arenicola may be considered as vulnerable. On the other hand, by applying the IUCN Red List Criteria (IUCN 2012), C. arenicola falls in the category of Endangered (EN) and meets the following criteria: A2c, as much as 50% decline of the population during the next 50 years inferred by the decline in habitat quality in the area of occupancy; B2c(ii,iii,iv), at present the area of occupancy is estimated as less than 500 km 2 , moreover, there are likely to be extreme fl uctuations in the area of occupancy because of its unstable habitat. Paratypes

Discussion
Although, the phylogenetic relationships of Cissampelos and the other genera in the Cissampelinae are still unresolved, Cissampelos is the oldest name in the clade and will therefore stand any future generic reassessment. While Cissampelos arenicola has not yet been included in any DNA based studies and as a result its phylogenetic affi nities are not known, and despite the fact that the sepals and petals are variable in number, they are consistently located on the adaxial side of the carpel, a feature that is shared by all other studied species of Cissampelos with exception of C. capensis. Hence, we are confi dent that our new species belongs to Cissampelos s. str.
Th e genus Cissampelos is characterized by its dioecy, as are nearly all Menispermaceae. However, rare instances of monoecy have been reported by Miers (1871), and we observed monoecy in two collections (Nee 48487; 51401) both from Bolivia, in which a few pistillate fl owers and immature fruits were observed in the same infl orescence that is predominantly staminate. Petals of pistillate fl owers in this infl orescence resemble these of staminate fl owers in being cupuliform. Th ere are reports of sex switching in Menispermaceae such as Asian Tinospora cordifolia (Willd.) Miers, where complete switching in a plant from staminate to hermaphrodite and back to staminate fl owers have been observed (Geetha et al. 2007). Only plants with staminate fl owers exhibit this labile sex expression, while plants with pistillate fl owers show consistency in their expression (Geetha et al. 2007, Malpotra et al. 2009. Th e report of monoecy in the American species Disciphania spadicea Barneby is based on observations of a single collection from Jalisco (Mexico) (Carrillo-Reyes et al. 2013) which had both staminate and pistillate infl orescences on the same plant. Our case is similar to that of Tinospora where infl orescences with mostly staminate fl owers may have a few pistillate fl owers.
Anther cells in C. arenicola vary from 6-10 (Table 1), with about 45% of the sampled 24 staminate fl owers having an 8-locular synandrium. Th is condition is also rarely observed in Cissampelos grandifolia Triana & Planch., but is unknown in the remaining American species of Cissampelos recognized by Rhodes (1975), which all have a 4-, rarely 6-locular synandrium. Similar 8-locular synandria are most commonly found in the vining C. mucronata A. Rich. from tropical Africa. Cissampelos mucronata diff ers from C. arenicola, however, in several characters, most obviously, larger leaves that are densely light golden hispidulous-tomentose on both surfaces. Vegetatively, Cissampelos arenicola superfi cially resembles the southern African Cissampelos capensis, from which it can be distinguished by the staminate fl owers with a 6-10-locular (vs. 4-locular) synandrium, and pistillate fl owers having sepals and petals located at the adaxial side of the carpel only. Th e larger and scarcely ornamented endocarp of C. arenicola (Fig.  3A) is readily distinguished from both, C. capensis (4.2 × 5.5 mm), and C. mucronata ( Fig. 3B-C), (4.3 × 4.7 mm), respectively (Table 1).