The systematics of the worldwide endoparasite family Apodanthaceae (Cucurbitales), with a key, a map, and color photos of most species

Abstract Using morphological, nuclear, and mitochondrial data, we here revise the taxonomy of Apodanthaceae and allocate the 36 names published in the family to ten biological species in two genera, Apodanthes and Pilostyles. All species are endo-parasites that live permanently inside trees or shrubs of the families Salicaceae or Fabaceae and that only emerge to flower. Because of this life history, Apodanthaceae are among the least known families of flowering plants. Nevertheless, the World’s herbaria as of 2013 hold at least 785 collections that, in combination with DNA phylogenies, permit well-founded species circumscriptions and geographic range maps. We also provide a key to all species, discuss the newly accepted or synonymized names, and make available color photos of six of the ten species.


Introduction
Apodanthaceae Tiegh. ex Takht. (Cucurbitales) is a family of endoparasites that live entirely in their host's stems and only become visible once the strictly unisexual fl owers have burst through the bark. Th is life style, added to the small size of the fl owers and patchy occurrence of the apparently mostly dioecious populations, has made it diffi cult to collect good and complete herbarium material (including both sexes and fl owering and fruiting specimens). While populations once identifi ed may be recollected at the same time year after year, usually only local botanists will have the opportunity to carry out such recollections. Apodanthaceae are disjunctly distributed in North and South America, mainland Africa, Iran, and Australia. Th ey occur in arid as well as humid tropical environments. Two genera have been validly described, the worldwide Pilostyles , and Apodanthes from Central and South America.
Th e taxonomy of the genera and species of Apodanthaceae has not been studied since the work of Ida de Vattimo-Gil (Vattimo-Gil 1950, 1955, 1971, 1973. Modern molecular-phylogenetic work based on representatives of most of the so-far named species (Bellot and Renner in review), together with study of collections deposited in the World's herbaria since the end of the 19th century, has revealed the need to synonymize many superfl uous names, a task that we carry out here. We also up-date the circumscription of the family and its two genera, and clarify that they have specialized on diff erent hosts, namely Salicaceae (mainly Casearia ) and Fabaceae.
To achieve a better understanding of species boundaries and relationships, and to clarify the species' geographic and host ranges, we compared loans from numerous herbaria, dissected fl owers, and isolated DNA from multiple collections. Molecular markers useful for these obligatory holoparasites are the nuclear 18S ribosomal RNA region and mitochondrial matR (Barkman et al. 2004;Bellot and Renner in review), and we show here that these markers can be used to place incomplete collections (for example, those of only one sex or only with fruits) in the correct species. Lastly, we provide an annotated key to all species that we recognize, and brief descriptions of their diagnostic traits along with color images and comments on their geographic and host ranges.

Plant material, DNA extraction and sequencing, phylogenetic analyses
We enlarged the DNA data matrix of Bellot and Renner (in review) by extracting DNA from additional specimens representing either unusual individuals or potential new species. No DNA sequences could be obtained from P. stawiarskii , known only from two collections in R, and P. holtzii, the only collection of which was destroyed in World War II. Suppl. material 1 shows species names and their authors, herbarium vouchers, and GenBank accession numbers. In total, 10 sequences (3 of 18S and 7 of matR ) were newly generated for this study.
Total genomic DNA was extracted from herbarium specimens using the commercial plant DNA extraction Invisorb® Spin Plant Mini Kit (Stratec molecular, Berlin, Germany). Th e mitochondrial matR and the nuclear 18S genes were amplifi ed using the primers listed in Bellot and Renner (in review). PCR products were purifi ed with the ExoSAP or FastAP clean-up kits (Fermentas Life Sciences, St. Leon-Rot, Germa-ny), and sequencing relied on the Big Dye Terminator v. 3.1 cycle sequencing kit (Applied Biosystems, Foster City, CA, USA) and an ABI 3130-4 automated capillary sequencer. Chromatograms were checked and sequences were edited using Geneious R7 (Biomatters, available from http://www.geneious.com), and contigs were then blasted against GenBank to rule out contamination. Alignments of the clean sequences were performed using the program MAFFT v. 7 (Katoh 2013) resulting in matrices of 1626 and 1727 aligned nucleotides for matR and 18S, respectively. We failed to amplify the gene matR from the African Pilostyles aethiopica and from the Iranian P. haussknechtii . Phylogenetic reconstructions relied on maximum likelihood (ML) as implemented in RAxML-7.2.8-ALPHA (Stamatakis 2006), using the GTR + G model of nucleotide substitution with 100 bootstrap replicates under the same model. Trees were rooted on Corynocarpus laevigatus (Corynocarpaceae; Cucurbitales), based on Filipowicz and Renner (2010).

Morphological data and assessment of the host ranges of Apodanthaceae
We geo-referenced locality data from 785 herbarium collections on loan from the herbaria B, G, C, GH, K, M, MO, MSB, W, NA, PMA, and SI and added data from the Global Biodiversity Information Facility (GBIF Backbone Taxonomy, 2013-07-01, http://www.gbif.org/species/7279680). We also recorded host names, up-dating their taxonomy as relevant. All label information was compiled in a database using the Botanical Research and Herbarium Management System (BRAHMS, http://herbaria. plants.ox.ac.uk/), and maps were produced using DIVA-GIS 7.5 (http://www.diva-gis. org). Collections were sorted by geography, fl owering specimens were sexed to evaluate sexual dimorphism, and a representative number of fl owers were then dissected under a stereoscope. For each dissected fl ower, the fi rst author recorded the number, arrangement and size of the tepals, shape and ornament of the pistil/central column, number of pollen sacs, presence of hairs and presence of a nectary at the base of the fl ower. Pictures of representative organs were taken using a Dino-Lite USB microscope model AM413ZT (Dino-Lite Europe) and the DinoCapture Imaging software version 2.0 of the same company.

Genus and species boundaries in Apodanthaceae
Th e dissections showed that species have characteristic fl ower sizes, number of tepals, tepal cilia, and number of anthers rings. For the American species, we use these differences in the key (below). Suppl. material 2 shows measurements and counts from the 123 dissected fl owers. Six collections could not reliably be assigned to these groups because their fl owers were slightly unusual: R. Callejas (Cannabaceae), but a partial matR sequence of this host BLASTed to Casearia nitida , making it likely that the host was in fact a Casearia . If that is the case, this would suggest that the collection represents an Apodanthes . Th e few male fl owers of Apodanthes caseariae that have so far been dissected (Suppl. material 2) do not allow assessing the full morphological variability of the male fl owers of this species. Th erefore we had to rely on DNA for identifi cation. In terms of its matR (Fig. 1A) R. Callejas et al. 8062 was embedded among other sequences of A. caseariae , while in terms of its 18S (Fig. 1B), it was sister to them. We identifi ed the specimen as A. caseariae . Other matR and 18S sequences in the A. caseariae clade are from the type of the name A. minarum ( Mexia 4540 ) from Brazil. Its host was a Casearia and its (female) fl owers match those of A. caseariae (Suppl. material 2). We therefore synonymize A. minarum under A. caseariae (an action carried out below).
In combination, the present morphological and molecular results show that Apodanthaceae comprise at least ten biological species that can be allocated to two mutually monophyletic genera. In the Americas, these are Pilostyles thurberi in the southern United States of America and Mexico, P. mexicana in Mexico, Guatemala and Honduras, the widely distributed P. blanchetii from Panama to Jamaica to Brazil and Uruguay, and P. berteroi in Chile and Argentina. Th e Americas also harbor A. caseariae from Guatemala to Brazil (Fig. 2). Australia has three species, P. coccoidea , P. collina , and P. hamiltonii ; Iran has P. haussknechtii, and Africa has P. aethiopica . Th e second African species, P. holtzii has not been recollected since 1907, when its type collection was made. Another species, the southern Brazilian P. stawiarskii, is only known from two specimens (one of them the type) collected at the same locality in Jan. /Feb. 1948 andDec. 1949; morphologically it resembles P. blanchetii . Th e host ranges of our accepted genera and species do not overlap. Apodanthes parasitizes only Salicaceae, whereas Pilostyles parasitizes only Fabaceae. As seen on Figure 3, there is a correspondence, although not perfect, between the phylogenies of host genera and parasitic species, and host specialization may have played a role in speciation of Apodanthaceae. At the species level, Table 1 shows that species of Apodanthaceae can grow on one or up to thirteen host species. As seen in Figures 2 and  3, our species concepts are corroborated by geographic and host ranges, except in the   case of Apodanthes caseariae and Pilostyles blanchetii , both widespread in Brazil. Th ese two species have diff erent sized fl owers (see below), and parasitize phylogenetically distantly related hosts (Fig. 3).

Description of the family
Stem-endoparasites, non-photosynthetic. No leaves, stem or roots, instead an endophytic system of cells inside the stem parenchyma of the host, fl owers bursting    with radial symmetry. Perianth composed of 2 or 3, rarely 4, whorls of tepals ( Fig.  4A-C), the latter sometimes with hairs along their margins (Fig. 4D), or a hair cushion at their basis (Blarer et al. 2004). In male fl owers, the staminal fi laments completely fused and forming a tube around a central column that is usually fused to the column (Fig. 5A, D), the up to 72 pollen sacs arranged in 1-4 rings around the column apex (Fig. 5A), the column apex dome-shaped and circled or covered by single-celled hairs (Fig. 5A, D), a basal nectar cushion in both sexes (Fig. 5E). Female fl owers without staminodes and with a single thick style topped by the dome-shaped stigma (Fig. 5B, C, E). Ovary semi-inferior, placentation parietal with 50-300 ovules (Fig. 5C, E). Seeds ca. 0.5 mm long (Bouman and Meijer 1994). Fruit a fl eshy berry.
Key to the genera and species of Apodanthaceae

Note on a possible new species of Pilostyles
Flavio González and Natalia Pabón-Mora, at the university of Antioquia in Colombia, are studying the ecology and morphology of Apodanthaceae in Colombia (González and Pabón-Mora accepted a) and are describing a new species of Pilostyles (González and Pabón-Mora accepted b). Th is species is the fi rst Pilostyles parasitizing the legume genus Dalea in South America and occurs in dry valleys of the Colombian Eastern Cordillera at altitudes above 2000 m. Morphologically, the new species is most similar to P. berteroi , which grows in the Chilean and Peruvian Andes at up to 3000 m of altitude (Fig. 2) and parasitizes Adesmia (closely related to Dalea , see Fig. 3).

Conclusion
By combining morphological and molecular information, we show that Apodanthaceae comprise 10 species and that morphological distinctions fi t well with geographical disjunctions and specializations on diff erent hosts (Salicaceae vs. Fabaceae). DNA sequences of mitochondrial matR and nuclear 18S rDNA, along with morphology and geography permit identifying any collection of Apodanthaceae. A wider sampling of the morphological variation, especially of male Apodanthes caseariae and female Pilostyles blanchetii , P. mexicana and P. thurberi , however, is needed to determine whether some unusual specimens might deserve to be ranked as subspecies.