Croton calcareus: a new species of dragon’s blood (Euphorbiaceae) from dry forest in the state of Chiapas, Mexico

We describe Croton calcareus Riina & Mateo-Ram. sp. nov., a new species in Croton section Cyclostigma (dragon’s blood trees) from the state of Chiapas (Mexico). This species is a small tree growing in dry forest on calcareous substrates. Both morphological and molecular data support C. calcareus sp. nov. as a new species closely related to C. redolens, another dry forest taxon from northern Venezuela. We provide illustrations, a distribution map and suggestions for species conservation status. The new species along with Croton draco are the only known representatives of C. section Cyclostigma occurring in Mexico.


Introduction
Croton L. species popularly known as dragon's blood ('sangre de drago' or 'sangre de grado' in Spanish) belong to C. sect. Cyclostigma Griseb. This section is an arborescent clade of 48 species occurring mainly in lowland and montane forests along the Andes van Ee et al. 2011) and the Atlantic Rain Forest region in south-eastern Brazil (Santos et al. 2017;Farias et al. 2019). In the northern Neotropics, only one species in the section, Croton draco Schltdl., was previously known to reach Mexico.
As part of a revision of the common and widespread Croton draco, which occurs from Mexico to Panama, we noticed several specimens from Mexico that had obvious morphological differences with C. draco as well as differences in habitat. Further examination of these specimens revealed an undescribed taxon, which we assign here to C. sect. Cyclostigma based on its morphological affinities with species in this section and support from molecular phylogenetic evidence. countries (Jones 2003;Salatino et al. 2007). Species from sect. Cyclostigma occur predominantly in mesic forests Arévalo et al. 2017); however, a small number of species are restricted to seasonally dry forest vegetation in different areas of the Neotropics, such as C. churutensis Riina & Cornejo (Riina et al. 2007) in Ecuador, C. tumbesinus Riina (Feio et al. 2018b) in Peru, C. redolens Pittier in northern Venezuela and C. charaguensis Standl. in Bolivia. In this paper, we describe and illustrate a new species from the state of Chiapas (Mexico), which adds to the list of dry forest specialists of this diverse and medicinally important Neotropical Croton clade.
For the phylogenetic analysis, we used a subset of the nuclear rDNA internal transcribed spacers (ITS) dataset from Riina et al. (2009), including species of sect. Cyclostigma sampled there, a selection of other Croton sections and one outgroup (Brasiliocroton mamoninha P.E.Berry & Cordeiro). To this dataset, we added two newly generated sequences of the new species, C. calcareus Riina & Mateo-Ram. sp. nov., and two sequences of C. draco from GenBank (https://www.ncbi.nlm.nih.gov/genbank/). GenBank accession numbers of all the sequences used in the analysis are shown in Table 1. DNA extraction, amplification and sequencing of ITS followed the same laboratory procedures as in previous Croton phylogenetic studies van Ee et al. 2011). Sequences were aligned manually using Mesquite ver. 3.2 (Maddison & Maddison 2017). The aligned matrix, in fasta format, is provided as Supplementary file 1.
The phylogenetic analysis was performed in MrBayes 3.2.6 (Ronquist et al. 2012) using the GTR+I+G substitution model as estimated with jModelTest 2.1.6 (Darriba et al. 2012) using the Akaike Information Criterion (AIC). The analysis included two runs of 10 million generations in MrBayes, sampling trees every 1000 generations. One-fourth (25%) of the MCMC samples from each run was discarded as ʻburn-inʼ. Tracer ver. 1.7.1 (Rambaut et al. 2014) was used to determine run convergence and stationarity as indicated by the effective sample size (ESS) values, which should be higher than 200. Node posterior probability (PP) values were obtained by computing a 50% majority rule consensus of the post-burn-in trees from both MCMC chains using the sumt command. We used FigTree ver. 1.3.1 (Rambaut 2006) to visualize and edit the consensus tree.
We ran a preliminary analysis with all the Cyclostigma accessions from Riina et al. (2009) to assess the phylogenetic position of C. calcareus sp. nov. within the section (data not shown). Based on this preliminary phylogeny, we reduced the sampling within the Cyclostigma clade to exclude taxa distantly related from C. calcareus sp. nov. (i.e., all the Brazilian species from the Atlantic Rain forest and most species from the south-central Andes). We ran the final Bayesian analysis on the reduced dataset (Table 1) following the steps indicated above.
The species distribution map was produced using R software (https://www.r-project.org) and the dismo package (Hijmans et al. 2010). Only two of the seven available collections had geographic coordinates on their labels, so we estimated coordinates for the remainder with the locality information using Google Earth. The proposed conservation status was based on the criteria of the International Union for the Conservation of Nature Guidelines (IUCN 2012). Extent of occurrence (EOO) was assessed using GeoCat (Bachman et al. 2011).  has been confused with C. draco Schltdl., another dragon's blood from Mexico, but it differs from this species in having shorter inflorescences (8-10 cm vs 15-43 cm in C. draco) and larger fruits (only 4-7 × 4-7 mm in C. draco).

Etymology
The specific epithet refers to the calcareous substrates where the species occurs.

Phylogenetic placement and relationships
The ITS alignment consisted of 686 aligned positions. The resulting tree topology (Fig. 1) was congruent with previous analyses of Croton using the ITS region for the clades involved van Ee et al. 2011). This phylogeny indicates that Croton calcareus sp. nov. is indeed a member of Croton sect. Cyclostigma and that it is most closely related to C. redolens and C. gossypiifolius Vahl (Fig. 1). The two sequences of C. calcareus sp. nov. are identical and share two unique positions with respect to the rest of sequences in the matrix. The accessions of Mexican species with which C. calcareus sp. nov. has been confused in the past (i.e., C. draco, C. xalapensis Kunth and C. verapazensis Donn.Sm.) are indicated with an asterisk in the phylogeny (Fig. 1). Of these, C. draco is the most closely related to the new species, coming out in the same clade (sect. Cyclostigma), the other two species belong to two different sections/clades of Croton (Fig. 1).

Distribution, ecology and phenology
Croton calcareus sp. nov. appears to be restricted to the state of Chiapas (Mexico) where it grows on limestone outcrops, in low deciduous forests on rocky slopes, steep river canyons and river floodplains, between 550-1500 m elevation (Fig. 4). Flowering between August and February and probably fruiting between March and July.

Conservation status
The species could be categorized as Vulnerable (VU D2; IUCN 2012). The extent of occurrence of Croton calcareus sp. nov. is only 2718 km 2 and it is only known from four different localities in two notadjacent municipalities. The species appears to be under low human pressure since three of the known localities (Municipality of Ocozocoautla) are within the buffer zone of the Selva el Ocote Biosphere Reserve (Ochoa-Gaona 1996; UNESCO 2012). Most of the known localities are of limited access due to their vicinity to limestone rocky formations along rivers.

Discussion
Our phylogenetic analysis supports Croton calcareus sp. nov. as a distinct lineage in C. sect. Cyclostigma sensu van Ee et al. (2011), which, combined with the observed distinct morphological features, warrants recognition at the species level. Based on the ITS phylogeny and morphology, the new species is most closely related to C. redolens, a dendroid shrub (up to 3 m tall) occurring in dry forest vegetation associated with rocky outcrops in northern Venezuela. Croton calcareus sp. nov. also belongs to the same clade that includes C. draco, the only other species in sect. Cyclostigma known to occur in Mexico. Characters shared among C. calcareus sp. nov., C. draco, C. redolens and other members of sect. Cyclostigma include, but are not limited to, arborescent habit, indumentum of stellate trichomes, red latex, conspicuous stipules, petiolar nectary glands, bisexual cymules at the lower portion of the inflorescence axis, bifid styles and more than 15 stamens. For this reason, it is not surprising that specimens of C. calcareus sp. nov. have been confused with C. draco in the past.
Among the many morphological differences between C. calcareus sp. nov. and both C. redolens and C. draco (Table 2), the most obvious ones are fruit size and inflorescence length; although the latter is only relevant in the case of C. draco. Inflorescences are much longer in C. draco and fruits are much larger in C. calcareus sp. nov. (Table 2). Unfortunately, only one of the examined collections (Hampshire et al. 1161, the type) had fully mature fruits and seeds. Plant stature and habitat preference are also important features setting the two Mexican species ( C. draco and C. calcareus sp. nov.) apart (Table 2).
There are also differences, although with some overlap, in the leaves among the three species in question.
In Croton calcareus sp. nov. leaves can have a rounded or cordate base ( Fig. 1C-D) and acute to obtuse apex, whereas in C. redolens the base is mostly rounded and rarely cordate and the apex is acute, and in C. draco the base of the leaves is usually cordate and the apex acuminate (Table 2). In addition, the texture of the adaxial leaf surface is soft to touch in C. draco and C. redolens, while in C. calcareus sp. nov. it has a rough texture. Stipules are usually informative distinguishing species within sect. Cyclostigma Farias et al. 2019). In Croton calcareus sp. nov., there is variation in the shape of stipules depending on their state of development. When young, stipules in C. calcareus sp. nov. are triangular in shape and they become more linear and much longer as they grow (Fig. 1B), whereas in C. redolens and C. draco young and old stipules have more or less the same shape (Table 2).  (Fig. 1). Croton xalapensis is a shrub in C. sect. Adenophylli Griseb., the largest section of Croton and the sister clade of sect. Cyclostigma (van Ee et al. 2011). It is also one of the few sections of Croton with a distinct synapomorphy, i.e., the presence of three inflated lobes at the apex of the fruit columella van Ee et al. 2011). So, C. xalapensis can be easily distinguished from C. calcareus sp. nov. by its shrubby habit and its trilobed columella apex. Croton verapazensis, from Central America, is superficially similar to C. calcareus sp. nov. and it also grows in dry forest. However, it is member of a different clade of Neotropical Croton, namely C. sect. Corylocroton G.L.Webster, and can be distinguished from any member of sect. Cyclostigma, including C. calcareus sp. nov., mainly by its unique petiolar nectary gland morphology (apical part with almost the same diamater as the stipe; in sect. Cyclostigma it is much wider than the stipe), presence of stellate-lepidote trichomes, leaf margins serrate to coarsely dentate and presence of unisexual cymules at the base of the inflorescence axis. Finally, as in C. draco, both C. xalapensis and C. verapazensis also have much longer mature inflorescences and smaller capsules and seeds than C. calcareus sp. nov.

Croton draco Croton redolens
Stipules Short triangular to long filiform Subulate to broadly subulate