Melothria domingensis (Cucurbitaceae), an endangered Caribbean endemic, is a Cayaponia

Abstract The Neotropical genus Melothria (Benincaseae, Cucurbitaceae) is a small group of yellow- or white-flowered climbers with small to medium-sized fruits. In 1899, Alfred Cogniaux described a species from montane rainforest in Haiti as Melothria domingensis, presumably based on the overall similarity in habit, leaf shape, and fruit morphology of his incomplete herbarium material to other Central American Melothria species. Melothria domingensis is still rare in European and American herbaria and the species has never been studied in detail. We here present molecular and morphological analyses, which show that the species is misplaced in Melothria and instead belongs in the distantly related tribe Cucurbiteae in the genus Cayaponia. We illustrate the species, provide the formal transfer and an extended description, and discuss the phylogenetic, biogeographic and ecological implications, including the finding that most likely bee- and not bat-pollination is ancestral in Cayaponia.


Introduction
Th e genus Melothria L. (including Melancium Naudin, Cucumeropsis Naudin, and Posadaea Cogn.) includes 12-15 species, confi ned to arid plains, clearings and forest margins, grass-or woodlands from the southern United States through Central and South America down to northern Argentina (Schaefer and Renner 2011 a, b). One species, Melothria sphaerocarpa (Cogn.) H.Schaef. & S.S.Renner, is also found in tropi-cal West Africa, where it might have arrived only recently through human-mediated transport (Schaefer and Renner 2010); and another species, Melothria pendula L., is locally invasive in tropical Asia (De Wilde and Duyfj es 2010). Melothria species are monoecious, small to medium-sized, herbaceous climbers with usually simple leaves, small yellow or white fl owers, and the fruit a smooth, and often fl eshy berry to 20 cm in M. sphaerocarpa. Each fruit contains numerous, strongly compressed seeds; and the testa tends to be smooth, white, and often covered by long appressed hairs (Schaefer and Renner 2011b).
In 1899, Alfred Cogniaux described a new species of Melothria based on herbarium material from the Caribbean island of Hispaniola (Haiti and the Dominican Republic), which he had received from the Krug and Urban collection (Urban 1899). Th e type material (L. Picarda 1503, BR) contains complete leaves and fruit plus seeds but no fully developed fl owers. While the overall habit, leaf shape and fruit type are quite similar to Central American species of Melothria, the ovoid seeds would be unique in that genus and are very similar to another, very distantly related genus: Cayaponia Silva Manso in the tribe Cucurbiteae. And indeed, the Brazilian Cayaponia expert Vera Gomes-Klein annotated a specimen of Melothria domingensis Cogn. in the Berlin Herbarium (E. L. Ekman 1319, B) as "Cayaponia sp." in July 1992; and two collections at New York Botanical Garden (P. Acevedo-Rodríguez et al. 13011 and 13274, NY) had originally been identifi ed as Cayaponia racemosa (Mill.) Cogn. by M. T. Strong in 2010. Th e aim of our study here is to test this hypothesis using molecular and morphological data and to fi nd out whether Melothria domingensis is indeed a misplaced Cayaponia or alternatively represents a unique type of seed morphology in Melothria.

Materials and method
Molecular analyses. Total genomic DNA was isolated from leaf samples of four herbarium specimens of Melothria domingensis (all collected in the Dominican Republic, Table 1) using a commercial plant DNA extraction kit (NucleoSpin, MACHEREY-NAGEL, Düren, Germany), and following the manufacturer's manual. Polymerase chain reactions (PCR) following standard procedures were used to amplify the entire nuclear ribosomal ITS region plus the following fi ve chloroplast regions: rbcL gene, trnL intron, and the three intergenic spacer regions trnL-trnF, rpl20-rps12, and trnH-psbA. PCR protocols and primers are given in Kocyan et al. (2007) and Schaefer et al. (2009). In addition, we used the primer pair trnH (5'-CGC GCA TGG TGG ATT CAC AAA TC) and psbA (5'-GTT ATG CAT GAA CGT AAT GCT C) designed by Sang et al. (1997) with an annealing temperature of 48°C to amplify the trnH-psbA spacer region. Crude PCR products were sent to Functional Biosciences, Inc. (Madison, WI, USA) for ExoSap cleaning and Sanger sequencing with the same primers used for PCR reactions. Seventeen sequences were newly generated for Melothria domingensis plus eleven ITS sequences for selected species of the genera Abobra Naudin, Calycophysum H. Karst & Triana, Cionosicys Griseb., Schizocarpum Schrad., and Tecunumania Standl. & Steyerm., all of the tribe Cucurbiteae, to obtain a more  balanced sampling in our ITS matrix (for GenBank accession numbers and vouchers see table 1). Raw sequences were edited with Sequencher 4.9 (Gene Codes, Ann Arbor, Michigan, USA) and aligned by eye, using MacClade 4.08 (Maddison and Maddison 2003). We then added to those alignments sequences for all Cayaponia species available on Genbank (mainly from Kocyan et al. (2007), Schaefer et al. (2009), and Duchén and Renner (2010)) plus a set of Cucurbitaceae species representing all genera of the tribe Cucurbiteae, three representatives of Melothria and representatives of the genera containing Caribbean species based on Schaefer et al. (2008). Bayesian and maximum likelihood analyses were performed with a fi nal dataset of 67 accessions representing 60 species. Data matrix and trees have been deposited in TreeBASE (http://www.treebase. org/) study number S13322. Maximum likelihood (ML) analyses and non-parametric bootstrap searches (BS) with the fast-bootstrap algorithm were performed using RAxML-VI-HPC v. XX (Stamatakis et al. 2008). RAxML searches relied on the GTR + G + I model and model parameters were estimated over the duration of specifi ed runs. Bayesian inference also used the GTR + G model (with the default four rate categories) and relied on MrBayes v. 3.2.1 x64 (Ronquist and Huelsenbeck 2003). We analyzed the combined dataset with two partitions (plastid and nuclear ITS), allowing partition models to vary by unlinking gamma shapes, transition matrices, and proportions of invariable sites. Markov chain Monte Carlo (MCMC) runs started from independent random trees, were repeated twice, and extended for ten million generations, with trees sampled every 1000th generation. Convergence was assessed using Tracer 1.5 (Rambaut and Drummond 2003). Trees saved prior to convergence were discarded as burn-in (2000 trees), and a consensus tree was built from the remaining trees.
Morphological analyses. We studied Melothria domingensis specimens from the following herbaria: BR, GH, NY, U, and US. For comparison, both authors also studied a large number of Cayaponia and other Neotropical Cucurbitaceae specimens from all major European and American herbaria over the past decade. All measurements given in the text are from dry herbarium specimens.
Analysis of pollination syndrome evolution and biogeography. We used the same approach as described in Duchén and Renner (2010), namely ancestral character state reconstruction under maximum likelihood in Mesquite v. 2.72 (Maddison and Maddison 2009) based on the Markov k-state one-parameter model. We added Melothria domingensis to the matrix of that previous study with the character states "bee pollination" (based on fl ower morphology) and "rainforest habitat" (based on herbarium label information).

Molecular analyses
Analyses of the nuclear ITS data matrix (55 accessions, 984 aligned nucleotides) and the individual and combined plastid matrices (67 accessions, 4831 aligned nucleotides) produced congruent phylogeny estimates (Fig. 2, Fig. 3; phylogenies for individual plastid marker not shown), with all areas of discordance being restricted to branches with low support (i.e., BS <60% and Bayesian posterior probability (PP) <0.9). We therefore combined the ITS and plastid matrices into a single matrix (68 accessions, 60 species, 5673 aligned nucleotides) and in the following focus in our discussion on the phylogeny estimate built using this largest matrix (Fig. 4)

Morphological comparison
Comparison of seed, fl oral, fruit, and vegetative characters of Melothria domingensis with all available Cayaponia, Cionosicys, and Melothria material reveals that seed number, size and shape are most similar to Cayaponia and not to Melothria or Cionosicys. Mottled fruits like those of Melothria domingensis also occur in Cionosicys, but all currently known species of Cionosicys have much larger fruits and are many-seeded. In Melothria, fruits can be small and striped or mottled but all currently known species of that genus have many-seeded fruits with strongly compressed seeds. Only in Cayaponia do we fi nd small fruits with few, tumid seeds.
Within Cayaponia, the relatively small leaves and few-fl owered fascicles of Melothria domingensis are most similar to Cayaponia quinqueloba, an endemic from the southeastern United States. Th e fruits of C. quinqueloba, however, have the typical coriaceous or chartaceous exocarp of many other Cayaponia species, and not the distinctive spots of Melothria domingensis fruits. Another Cayaponia species with mottled fruits is Cayaponia tibiricae (Naudin) Cogn. (syn. C. martiana (Cogn.) Cogn.) of eastern Brazil, but its infl orescence is branched like that of C. racemosa (Mill.) Cogn. and a few others.
Distribution. Endemic to the Greater Antilles, island of Hispaniola, and perhaps Puerto Rico (one record from Ponce, Toro Negro Commonwealth Forest, between Cerro Maravillas and Monte Jayuya, along road off highway 143, 1190-1200 m, 26 Feb 1993, Breckon et al. 4427, NY, specimen not seen).
Conservation status. Listed as in danger of extinction ("peligro de extinción") on an informal webpage for the fl ora and fauna of Hispaniola (Marcano 2009). Th e available information on distribution, population size, and threats does not seem suffi cient for a formal classifi cation following IUCN red list criteria.

Pollination syndrome evolution
Our re-analysis of the evolution of pollination syndromes in Cayaponia reveals that bee pollination is most likely the ancestral state for the clade (Fig. 5) followed by one shift to bat pollination along the stem of the C. tubulosa Cogn.-C. prunifera (Poepp. & Endl.) P.Duchen & S.S.Renner clade and one reversal to bee pollination within the same clade (C. espelina (Silva Manso) Cogn.). A third shift is inferred for C. pilosa Cogn., which is bat pollinated but nested in a bee pollinated clade (Fig. 5).

Ancestral habitat reconstruction
We fi nd that the ancestral habitat of the Cayaponia lineage was most likely rainforest, which is in agreement with most extant species still being confi ned to some type of rainforest habitat (Fig. 6). Only few species, including C. espelina, C. pilosa, and some members of the clade containing C. attenuata (Hook. & Arn.) Cogn. shifted to more open habitats (Fig. 6).

Discussion
Molecular and morphological evidence both show that Melothria domingensis is best placed in the Cayaponia clade and probably close to the North American C. quinque-loba. However, due to the poor resolution in parts of our Cayaponia phylogeny, we are unable to identify exact sister group relationships. Better taxon sampling and additional variable DNA markers are needed to solve this question.
Th e newly identifi ed Cayaponia species from Hispaniola is a rain-or cloud forest inhabitant with small, probably diurnal fl owers on short pedicels that match all characters for bee-pollination in Cayaponia discussed in Duchén and Renner (2010). It therefore does not fi t to the pattern of pollinator shifts from bat-to bee-pollination as an adaptation to open savanna habitats hypothesized for Cayaponia by Duchén and Renner (2010) based on an analysis including 19 of the c. 60 species of Cayaponia. And indeed, our re-analysis of the pollination syndrome evolution in Cayaponia also questions the fi nding that bat pollination might be the ancestral condition in the clade. We fi nd that based on the extended sampling, the most likely ancestral condition is bee pollination but with still 70% of the currently known Cayaponia species remaining to be sequenced, this pattern can easily change in the near future. We can confi rm, however, at least one inferred shift from bat-to bee-pollination in C. espelina, which would be one of very few transitions in that direction (Duchen and Renner 2010; Van der Niet and Johnson 2012). Fieldwork on pollinators of C. espelina, a common species of the Brazilian Cerrado, would be needed to elucidate this interesting case further.
Cayaponia domingensis is one of only few species in the genus and in the entire family Cucurbitaceae that occur above 2000 m a.s.l. (the highest collections known are from 2100 m a.s.l., Liogier & Liogier 25690 (NY), A. Liogier 13134 (NY) and 2201 m a.s.l., P. Acevedo-Rodríguez et al. 13011 (NY)). Th e mechanisms that allow this species to thrive at those altitudes are unknown but might be interesting to study. Most Cucurbitaceae are highly frost-sensitive and survive cold periods only as seeds (e.g. North American Sicyos angulatus L.), rootstocks (e.g. Central European Bryonia dioica Jacq.), or tubers (e.g. Th ladiantha dubia Bunge). Th e only perennial Cucurbitaceae that thrive at even higher altitudes are some Himalayan Th ladiantha species that reach up to 3500 m a.s.l.
Th e mottled fruits characteristic for Cayaponia domingensis are very rare in the genus and might have evolved as an adaptation to a specialised seed dispersal agent on the island. Unfortunately, we lack fi eld observations on dispersal agents and judging from fruit and seed size, a wide range of animals, including birds, lizards, and small mammals might be involved.
Recent transoceanic dispersal has been inferred for the ancestors of Cayaponia africana s.l. (Schaefer et al. 2009;Duchén and Renner 2010). While Duchén and Renner (2010) hypothesized a stepping-stone route via the Brazilian island Fernando de Noronha, our fi ndings suggest that long-distance dispersal out of the Caribbean might be another option.
Our results highlight that even after two decades of Molecular Systematics, we still need more sequencing combined with morphological analyses to sort out taxonomic problems in Cucurbitaceae and other understudied families. We can now be confi dent about the phylogenetic position of this enigmatic Caribbean endemic. Th is is, however, only a fi rst step and we now plan to do fi eldwork on Hispaniola to obtain accurate distribution data, fi nd out more about the ecology of this species, identify the threats to the remaining populations and ultimately develop a management plant to guarantee the survival of this unique endemic.