Phylogenetics of Leptocereus (Cactaceae) on Hispaniola: clarifying species limits in the L. weingartianus complex and a new species from the Sierra de Bahoruco

Abstract The Antillean genus Leptocereus represents an in-situ radiation among the Greater and Lesser Antilles of 19 currently recognized species. Extensive fieldwork carried out in the Dominican Republic over recent years has revealed that the species limits of Leptocereus of Hispaniola are more complex than previously thought. There are four currently recognized species that occur on the island, L. demissus, L. paniculatus, L. undulosus and L. weingartianus. We evaluate species limits in this group based on DNA sequence data and phylogenetic analysis, morphological characters and a survey of herbarium specimens from across Hispaniola. Based on our analyses, it is clear that at least five species occur on the island of Hispaniola, with the new species from Sierra de Bahoruco, L. velozianus, described here. We provide an identification key, distribution maps and photographic plates for all species on Hispaniola based on our own fieldwork and the study of herbarium specimens. The description of yet another species of Leptocereus on Hispaniola reiterates the importance of the poorly studied, but yet biodiverse, seasonally dry tropical forest in the Antilles.


introduction
Seasonally dry tropical forests (SDTF) are one of the most threatened forest types on the planet with only around 10% of their original coverage still remaining intact in the Neotropics (Banda-R. et al. 2016) mostly owing to anthropogenic pressures, such as charcoal production and agriculture (Pennington et al. 2000(Pennington et al. , 2004. This forest type is extensive throughout the Greater Antilles; however, it has been poorly studied (Gentry 1982;Pennington et al. 2005) compared to other forest types on the islands or dry forest from other parts of the Neotropics. Recent work has revealed that these understudied forests have much undocumented biodiversity (e.g., Mejía and García 1997;Fryxell and Clase 2007;Areces-Mallea 2017, 2018Majure et al. 2020;Martínez-Gordillo et al. 2020). Cactaceae are a conspicuous element of the diverse SDTF of the Greater Antilles, with roughly 94 taxa occurring in the region (Majure et al. unpubl. data). An estimated 35 species occur on the island of Hispaniola, including the Caribbean endemic Leptocereus (A. Berger) Britton & Rose. Leptocereus s.l. is a clade of 19 currently recognized species of trees and erect to sprawling shrubs, and represents an in-situ Antillean radiation that occurs in SDTF, primarily of the Greater Antilles, where it is most diverse, but also occurs in the Lesser Antilles on the island of Anegada (Barrios et al. 2020). Recent phylogenetic work by Barrios et al. (2020) showed that the two large tree species endemic to Cuba and Hispaniola, and traditionally circumscribed in the genus Dendrocereus (Britton and Rose 1920;Anderson 2001;Hunt et al. 2006), formed a monophyletic group [the Dendrocereus clade (D)] clearly nested within Leptocereus. Barrios et al. (2020) also resolved two other primary clades in Leptocereus, the Cuban (CU) clade consisting of species endemic to Cuba, and the Hispaniolan-Puerto Rican (EPR) clade consisting of taxa endemic to Hispaniola, Puerto Rico and outlying islands.
On Hispaniola, three species of Leptocereus (including Dendrocereus) were formerly recognized (L. paniculatus (Lam.) D. R. Hunt, L. undulosus (DC.) D. Barrios & Majure, and L. weingartianus E. Hartmann), and Areces-Mallea (2017) recently described a fourth species (L. demissus Areces) from southwestern Dominican Republic from the SDTF south of the Sierra de Bahoruco mountain range. Thus, currently, four species are recognized on Hispaniola. Barrios et al. (2020) showed that the Hispaniolan endemic, Leptocereus weingartianus, was sister to a clade containing another Hispaniolan endemic, L. paniculatus, and a clade of two Puerto Rican species, L. grantianus Britton and L. quadricostatus Britton & Rose. Leptocereus weingartianus is widely distributed across Hispaniola and is morphologically heterogeneous, forming relatively large shrubs or small trees (Areces-Mallea 2017) with apical branches that are often sprawling among surrounding, dense vegetation of SDTF (Majure et al. pers. obsv.). The new species, L. demissus, described by Areces-Mallea (2017), is morphologically very similar to L. weingartianus, especially considering the sprawling growth form of ultimate stem segments (Majure et al. pers. obsv.), however, several reproductive characters appear to separate the two species morphologically (Areces-Mallea 2017). Recent fieldwork in the Sierra de Bahoruco has revealed populations of an unidentified taxon of what morphologically appeared to be part of the L. weingartianus complex, however, which differed from typical L. weingartianus in stem and spine features. Therefore, it was clear given the morphological heterogeneity of L. weingartianus, and the wide distribution and phenetic similarity of putative close relatives, such as L. demissus, that phylogenetic analyses of these taxa on Hispaniola were greatly needed.
We wanted to determine whether the morphologically disparate populations from the Sierra de Bahoruco were indeed closely related to the L. weingartianus complex, and whether or not the morphologically similar L. demissus was distinct from L. weingartianus based on phylogenetic relationships. We sampled multiple populations of typical L. weingartianus, one population of the newly described L. demissus and several populations of the new morphotype from Sierra de Bahoruco, as well as taxa from all major clades of Leptocereus (sensu Barrios et al. 2020) and carried out a phylogenetic reconstruction based on nearly entire plastome sequencing from genome skimming. We also reviewed herbarium specimens from across the distribution of Leptocereus on Hispaniola, and we herein provide distribution maps, photographic plates, and an identification key to all species on Hispaniola, as well as a description of the new species uncovered during this work.

Materials and methods
All species of Leptocereus from Hispaniola, representing the EPR and D clades, as well as the new material from Sierra de Bahoruco, were sampled here for phylogeny reconstruction along with three taxa from the CU clade of Leptocereus and the Cuban L. nudiflorus (Britton & Rose) D.Barrios & S.Arias of the D clade. Likewise, L. grantianus and L. quadricostatus of the EPR clade were sampled. Outgroups included here were based on previous work by Barrios et al. (2020) and Majure et al. (unpubl. data) and included Armatocereus, Calymmanthium, Cereus, Melocactus, Selenicereus, and Stenocereus of Core Cactoideae (see Appendix 1).
Whole genomic DNAs of all taxa were extracted using a standard CTAB protocol with silica column cleaning (see Majure et al. 2019;Köhler et al. 2020). DNAs were resuspended in 300 ul of TE (Tris-EDTA) buffer (pH 8.0), and DNA quantity was analyzed on a Qubit 2.0 Fluorometer. Whole genomic DNAs were sent to Rapid Genomics LLC (http://rapid-genomics.com/home/; Gainesville, Florida, U.S.A.) for library preparation (including shearing) and sequencing via a genome skimming method, as in Majure et al. (2019). All taxa were sequenced on the Illumina HiSeq X platform using paired end reads (yielding 150 bp reads), and sixty samples were included per lane.
Raw reads of all taxa were imported in to Geneious (v. 11.1.5, Biomatters Ltd., Auckland, New Zeland) and reference-mapped using a previously, de-novo assembled partial plastome (including the large-single copy unit) of Melocactus pedernalensis M.M.Mejía & R.G.García (Majure, unpubl. data). Consensus sequences were then generated from reference-mapped plastomes, which were used for alignments.
Sequence alignment was carried out using the MAFFT (Katoh and Standley 2016) plugin in Geneious. We analyzed our 104,697 bp dataset (including indels) under maximum likelihood (ML) with the RAxML (Stamatakis 2014) plugin in Geneious using the rapid bootstrapping algorithm and undertaking 100 bootstrap pseudoreplicates.
Specimens (ca. 50) from multiple herbaria (JBSD, NY, S, US), as well as those generated from our own fieldwork (DES, FLAS, JBSD), were consulted for determining the morphological distinctiveness of the new material from the Sierra de Bahoruco, as compared to the phenetically similar taxa L. weingartianus and L. demissus. Those data also were used to determine the distribution of all species of Leptocereus across Hispaniola and to generate our identification key to the species (in part).

results
Leptocereus s.l. was resolved as monophyletic in our phylogenetic analysis, and all three principal subclades recovered by Barrios et al. (2020) were recovered here as well (i.e., CU, D, and EPR subclades)-all three subclades were mostly well-supported (CU, bs = 100%; D, bs = 84%, EPR, bs = 100%). The CU subclade was sister to a well-supported clade (bs = 100%) composed of the D and EPR subclades. Multiple accessions of the two species composing the D clade, L. undulosus and L. nudiflorus formed clades and were resolved as sister taxa in our topology, further demonstrating their phylogenetic distinctiveness. Within the EPR clade, a clade composed of L. demissus and the new species L. velozianus was sister to a clade composed of two subclades, the first formed by all accessions of L. weingartianus and the second by L. paniculatus, L. grantianus and L. quadricostatus. Thus, our results show that L. weingartianus is not closely related to the new species L. velozianus, but rather more closely related to the rest of the EPR clade. Likewise, although L. demissus appears to be phenetically more similar to L. weingartianus, it was more closely related to the new species L. velozianus. In all cases where multiple accessions were used per species, those species formed well-supported clades ( Fig. 1).
Leptocereus velozianus (Figs 2, 3), which is restricted to the northwestern part of the Sierra de Bahoruco (Fig. 4), is slightly phenetically similar to the Puerto Rican species L. quadricostatus, given the crenate rib margins and spiny pericarpels, although those two species are not close relatives (Fig. 1). Leptocereus velozianus is most phenetically similar to two other Hispaniolan species (L. weingartianus and L. demissus -Figs 5, 9), especially L. weingartianus, but differs from them based on the spine color (white or cream versus yellow to yellowish-red, although, younger spines in L. velozianus can be yellow-cream colored with darker tips, thus slightly overlapping with L. weingartianus), and the overall size of the joints (1.2-3.5 cm in diameter in the latter two species and 2.7-3.7 in L. velozianus). Leptocereus velozianus has conspicuously crenate margins, generally more so than either L. weingartianus or L. demissus, although this is a variable character (Areces-Mallea 2017). Although Areces-Mallea (2017) mentioned that L. demissus has straight rib margins between areoles and L. weingartianus is more crenate, we found that character to be variable in both, with L. weingartianus also sometimes having nearly straight rib margins between areoles (Figs 5B, 9A). In general, L. velozianus can be separated from the other two species, L. demissus and L. weingartianus, by a suite of morphological characters, as well as its phylogenetic relationships to the other taxa (Fig. 1). Leptocereus velozianus does not share any major morphological features with either L. paniculatus or L. undulosus (Figs 6, 7).
As far as is known, L. demissus and L. velozianus are the most restricted species on Hispaniola, with L. demissus restricted to lower elevation dogtooth limestone of Parque Nacional Jaragua and the surrounding area south of the Sierra de Bahoruco, while L. velozianus is restricted to well-developed tropical dry forest along the north slopes of the Sierra de Bahoruco (Fig. 4). Leptocereus paniculatus, L. undulosus, and L. weingartianus are much more widespread, with L. weingartianus occurring mostly on the north island from its eastern extremity near Cabo Engaño to its western extremity in Haiti on Gonave Island in elevations ranging from near sea level to 755 m in the Sierra Martín García, Dominican Republic (see Additional specimens examined). Leptocereus paniculatus is quite widespread across the island and is mostly found at lower elevations from -30 m on Isla Cabritos to around 400 m near Sierra Martín García, while L. undulosus is mostly restricted to areas near the coast in elevations ranging from near sea level to 245 m (Fig. 8, see also additional specimens examined).

Discussion
Our topology differs only slightly from Barrios et al. (2020) in that the CU clade was sister to the rest of Leptocereus here, whereas, in Barrios et al. (2020) the EPR clade was sister to the rest of Leptocereus. In both topologies, the D clade was nested within Leptocereus. Although it has been suggested that L. undulosus and L. nudiflorus may be conspecific (Anderson 2001), the multiple accessions sampled here clearly show them as genetically distinct entities, with multiple accessions of each forming clades, this likely being further driven by their reproductive isolation, being restricted to Hispaniola and Cuba, respectively. Morphological characters separating these two taxa are currently under investigation (D. Barrios unpubl. data).
Our phylogenetic results clearly demonstrate that the new species described here, L. velozianus, is genetically distinct from the more widespread L. weingartianus, and L. demissus, likewise, is more closely related to L. velozianus rather than the phenetically more similar L. weingartianus. Areces-Mallea (2017) mentioned that L. demissus is physically separated from L. weingartianus by the Sierra de Bahoruco. Our phylogenetic results and fieldwork support this idea, given that all L. weingartianus sampled were collected north of the Sierra de Bahoruco and form a well-supported clade, however, L. weingartianus does occur in the southern peninsula at nearly the same latitude as L. demissus south of Massif de la Hotte in Haiti based on the proposed neotype of L. weingartianus by Areces-Mallea (2017) from Cote le Fer. Interestingly, L. weingartianus grows alongside L. velozianus in the Sierra de Bahoruco; however, we have seen no evidence of hybridization between the two species. Ploidy, though, has not been examined in any species of Leptocereus from Hispaniola but is currently underway. Leptocereus weingartianus also occurs alongside L. paniculatus in populations near Jimaní and L. demissus occurs with L. undulosus in Parque Nacional Jaragua; however, we likewise have seen no putative hybrids among those species' pairs. We consider that phenology and perhaps pollinator differences could be driving the lack of hybridization in Leptocereus on Hispaniola (although we have very little information on specific pollinators of these species), and in other parts of their ranges, L. weingartianus appears to occupy different ecological niches than L. paniculatus. For example, in the Sierra Martín García, although both species occur there, L. paniculatus occurs at much lower elevations than L. weingartianus, which begins to be found around 400 + meters (Majure et al. pers. obvs.). So, ecology also could be driving species divergence in this group of close relatives.
Below, we provide a description of the new species, L. velozianus, as well as a taxonomic treatment with identification key, distribution maps and photographic plates of the other four species of Leptocereus on Hispaniola. Updated descriptions of the other four species will be presented elsewhere (Encarnación, in prep.).

Taxonomic treatment
Key to the species of Leptocereus on Hispaniola Diagnosis. Differing from both L. weingartianus and L. demissus by the white young spines (vs. yellowish spines) and larger stem diameter (up to 3.7 cm in diameter in L. velozianus). Differing from L. demissus by the erect, primary trunk rather than the sprawling growth form, and oblong hypanthium in L. velozianus rather than obconic hypanthium as in L. demissus.
Etymology. The specific epithet, "velozianus" is given honoring the Dominican botanist Alberto Veloz, who is the Head and Curator of the Herbarium JBSD of the "Dr. Rafael M. Moscoso" National Botanical Garden of Dominican Republic. For 27 years, Veloz has dedicated his life to the study of the Hispaniolan flora and has conducted extensive fieldwork across the island, with many collections from the Sierra de Bahoruco, where this new species was found. Together with other botanists he has collected over 10,000 specimens and has published several papers on the flora in national and international journals. His publications have included different approaches, such as floristics, taxonomy, ecology and conservation. Veloz has also contributed to the formation of young botanists by involving students as part of the staff in the herbarium JBSD and through fieldwork. Currently, the species is only known from the northwestern slope of the Sierra de Bahoruco, southwest of the town of Jimaní (Fig. 4). Given the extent of tropical dry forest in and around that area, it is very likely that more localities of this species will be found.
Phenology. Leptocereus velozianus has been collected in flower and immature fruit during May and with mature fruit in June, July and November. Thus, it appears likely that L. velozianus may flower over the early to mid-summer months with fruit ripening later in the year.  Conservation status. Formal evaluation of conservation status will be undertaken by Encarnación (in prep.) for L. velozianus based on further field work and demographic study. However, based on the currently known limited distribution of the species, the few numbers of individuals that have been observed, as well as anthropogenic activity near populations of the species, we consider that this species could be Near Threatened based on IUCN criteria. Further fieldwork will be essential for providing a comprehensive assessment of the conservation status of L. velozianus. Fortunately, the larger population known for this species is within the Sierra de Bahoruco National Park, so it is mostly protected from large-scale anthropogenic disturbances. Furthermore, the SDTF in that area is very well developed (i.e., non-fragmented) and should provide extra protection for the species. Notes. Although type material of L. demissus is reported to be at JBSD, HAJB, HNT, and NY (Areces-Mallea 2017), we have been unable to locate types at any of these institutions for comparative analysis for this work. Thus, we have based our knowledge on the morphological traits of this species using those characters given by Areces-Mallea (2017), geography, and the one collection from near Oviedo made by us (Majure 5972-see specimens examined; Fig. 5 (1785) and thus formed part of the protologue. This includes an illustration of a fragment of a stem segment, flower and fruit. Notes. Leptocereus paniculatus is consistently recovered as closely related to the L. quadricostatus/L. grantianus clade ( Fig. 1; see also Barrios et al. 2020), although the species is much more robust and treelike compared to the other two species. It is the  Notes. Although several authors have suggested that L. undulosus may be conspecific with L. nudiflorus of Cuba (Anderson 2001, Hunt et al. 2006, Barrios et al. (2020) showed that the two taxa were sister to one another and likewise were genetically divergent; however, they only sampled one accession of L. undulosus. We reaffirm that relationship here with one more sample of L. undulosus included in our phylogenetic analysis from a population near Barahona, east of the previous accession used in Barrios et al. (2020) from Parque Nacional Jaragua. Both samples form a strongly supported clade, sister to L. nudiflorus. More detailed morphological studies are necessary across the populations of L. undulosus on Hispaniola. Considering the distance between populations of the two (Cuba vs. Hispaniola), it is very likely that they are reproductively isolated. Thus, there seems to be no reason to consider these two taxa as conspecific. We anticipate that further morphological and genetic study will provide further support for the recognition of these two species.

Leptocereus paniculatus (Lam.) D. R. Hunt
Although not represented in our distribution map of L. undulosus, as there are no collections from that site, Île de la Tortue likely represents one of the larger populations of the species on Hispaniola (Peguero pers. obsv.). This reiterates the extensive fieldwork that still needs to be carried out to fully document populations of species of cacti with herbarium specimens from across the island. Cacti are often uncommonly collected because of the difficulty in preparing specimens, and thus, are generally poorly represented in herbaria (Majure et al. 2017).  Cereus weingartianus E. Hartmann Monatsschr. Kakteenk. 14: 155. 1904.

Type.
Haiti. Lectotype (designated by Barrios and Majure, in review). Photo of type material of L. weingartianus in Hartmann (1904).
Notes. There has been some confusion around the type of L. weingartianus -Areces-Mallea (2017)