Croton maranonensis : a new species of Euphorbiaceae from the tropical inter-Andean dry valleys

. We describe Croton maranonensis Riina & Martín-Muñoz sp. nov., a species in Croton section Julocroton (Mart.) G.L.Webster from the seasonally dry tropical forests and shrublands of the inter-Andean valleys. This species is a small shrub occurring along the Marañón river valley in Peru and similar dry areas in southern Ecuador. We surveyed morpho-anatomical characters of the new species and closely related taxa. To con ﬁ rm the placement of the new species in C . section Julocroton , we conducted a molecular phylogenetic analysis including three accessions of the new species and selected representatives of section Julocroton and related groups within Croton L. Micro-and macro-morphological evidence, and molecular data support C. maranonensis sp. nov. as an independent lineage within the C . section Julocroton clade. We compared the new species with morphologically similar species in the same section that also occur in the Andean region, including C. ﬂ avispicatus Rusby, C. triqueter Lam., and C. hondensis (H.Karst.) G.L.Webster.


Introduction
Croton section Julocroton (Mart.)G.L.Webster is a monophyletic group (Van Ee et al. 2011) that consists of 42 species including the new species here described.Species in this section are monoecious herbs or shrubs with usually dense stellate indumentum, leaves without basilaminar/petiolar nectary glands, highly congested bisexual inflorescences composed of staminate flowers with 10-12 stamens and pistillate flowers with laciniate and unequal sepals (Van Ee et al. 2011).The section is mainly distributed in tropical areas of South America, with southern Brazil and Paraguay as the main centers of diversity (Cordeiro 1990;Van Ee et al. 2011;Caruzo et al. 2022).Only one species, C. argenteus L., has expanded its range outside South America, occurring also in Central America, Mexico, southern United States and the Caribbean (Van Ee et al. 2011).
Croton maranonensis sp.nov.occurs in the seasonally dry forests of the tropical Andes biodiversity hotspot, the most species-rich among the world's hotspots (Myers et al. 2000).The new species has been extensively collected along the Marañón Valley and in a few localities in southern Ecuador.The Marañón Valley is part of the Central inter-Andean valleys floristic group identified by DRYFLOR (2016), along with 11 other floristic nuclei of dry forest vegetation in the Neotropics.The levels of plant endemism in the Marañón Valley is extremely high (Marcelo-Peña et al. 2016), with many new endemic species being described from this area in the last decade (Moura et al. 2013;Michelangeli & Ulloa Ulloa 2016;Esquerre-Ibañez 2017;Jara et al. 2020;Montesinos-Tubée et al. 2020;Wood & Uría 2021).This suggests that the diversity of Marañón's flora has been underestimated and that there are probably more new plant species waiting to be described (Särkinen et al. 2011).
In the last floristic survey of the Marañón Valley flora, Euphorbiaceae was recorded as the second family in number of species, after Leguminosae, and Croton and Senna Mill.were the most speciesrich genera with 12 and 11 species, respectively (Marcelo-Peña et al. 2016).When considering the entire area covered by the Central inter-Andean valleys floristic group (northern Peru and Ecuador;DRYFLOR 2016), Croton also stands out as the genus with the highest number of species and high levels of abundance in many of the forest plots measured so far (DRYFLOR 2016;Quintana et al. 2017).In some of these areas, Croton plants are the dominant woody elements of the landscape, acting as ecosystem engineers (Espinosa et al. 2019).
In this paper we add to the floristic knowledge of the Central inter-Andean valleys and the Neotropical dry forest by describing Croton maranonensis sp.nov., a taxon in the understudied C. section Julocroton.We used both morphological and anatomical data to characterize the species and to compare it to its closest relatives in the section.We also performed a molecular phylogenetic analysis based on the ITS region to test the placement of the new species in C. section Julocroton, as initially suggested by morphology.Finally, we propose a preliminary assessment of the conservation status of the new species.
For the phylogenetic analysis we used sequences from the ITS (Nuclear ribosomal internal transcribed spacers) region using a selection of species of C. subgenus Geiseleria A. DNA extraction, amplification, sequencing, and alignment followed the same procedures as in Masa-Iranzo et al. (2021).The aligned ITS matrix was trimmed on both ends to minimize missing data (the alignments, in fasta format, are provided as Supp.file 1).Phylogenetic analysis was performed in MrBayes ver.3.2.7 (Ronquist et al. 2012) applying the GTR+I+G model (Masa-Iranzo et al. (2021).We did 2 runs for 10 million generations sampling every 1000 th generation, monitoring the standard deviation of split frequencies (< 0.01) for assuring convergence.Tracer ver.1.7.2 (Rambaut et al. 2014) was used to evaluate the Markov chains.As burn-in, the first 25% of MCMC samples were discarded.Using the sumt command we obtained a Bayesian tree with posterior probability (PP) values by computing a majority rule consensus of the post-burn-in trees.We used FigTree ver.1.4.4 (Rambaut 2006) to edit the consensus tree.
A preliminary assessment of the conservation status, including estimation of the area of occupancy (AOO) and extent of occurrence (EOO) were conducted using the GeoCat software (Bachman et al. 2011) and the coordinates available from the examined herbarium collections of the new species.For those without coordinates, we estimated them with Google Earth using locality and habitat information provided in the collection labels.

Etymology
The specific epithet of the new species refers to Marañón river valley, the location where the majority of specimens of C. maranonensis sp.nov.have been collected.
Main micromorphological features of Croton maranonensis sp.nov.
Leaves are amphi-hypoestomatic, with paracytic stomata and epidermal cells exhibiting straight contour (Fig. 3A) on both faces.Simple trichomes and stipitate stellate porrect trichomes were present (Figs 3B-C); trichome stipes were shorter on the adaxial sides than on the abaxial side.Sometimes idioblasts were observed at the base of the radial cells of stellate trichomes (Fig. 3D, in detailed image).Sclereids, connecting both leaf sides across the mesophyll, were present at the base of stellate trichomes (Fig. 3E).Unicellular, glandular trichomes were relatively abundant on the abaxial side (Fig. 3C) and rare on the adaxial side (Fig. 3B).Glandular trichome had a knob-like appearance with a narrow base and a dilated distal portion (Figs 3F-G).Epidermal cells were tabular with mucilage content (Fig. 3F); the mesophyll is dorsiventral (Fig. 3F).The contour of the midrib is biconvex, with 2-3 layers of annular subepidermal collenchyma (Fig. 3D).
The bicolateral vascular system is organized in an open arch with a dorsal bundle (Fig. 3D).The blade margin is straight with non-continuous palisade parenchyma (Fig. 4A).The petiole has a flat-convex shape (Fig. 4B) and is covered by a uniseriate epidermis.Three to five subepidermal layers of annular collenchyma surround the entire petiole (Fig. 4B).The bicolateral vascular system is organized in a closed arch with the bundles separated by parenchyma (Fig. 4B).Both young and fully expanded leaves present standard colleters scattered along the blade margin (Figs 4A, C).Clusters of colleters were observed at the base of stipules (Fig. 4D) and on the basilaminar/acropetiolar region of the leaf (Fig. 4F), while only a single colleter was present at the stipule apex (Fig. 4E).Standard colleters consist of one-layer secretory palisade epidermis, and parenchyma, which are vascularized by xylem and phloem (Fig. 4A, E).Idioblasts with druse occur in the mesophyll (Fig. 3F), midrib, and petiole.Non-anastomosed articulated laticifers (Fig. 4G) were observed in shoots and fully expanded leaves, always associated with phloem, often exhibiting a Y-shaped form (Fig. 4H).Specimens of C. flavispicatus and C. triqueter had similar anatomical features (Fig. 4I), with the exception of the presence of a pair of accessory bundles in the petiole (Fig. 4J, Table 1), which was absent in C. maranonensis sp.nov.(Fig. 4B), and the absence of simple trichomes on the blade abaxial side.The presence of simple trichomes on the abaxial side of the blade (Fig. 4I) and the occurrence of idioblasts at the base of the radial cells of stellate trichomes of C. triqueter were the only anatomical differences observed between C. triqueter and C. flavispicatus.

Phylogenetic placement and relationships
The ITS matrix, with 63 aligned positions, consisted of 32 sequences representing 24 species of Croton, including 9 from the focal group (C.sect.Julocroton).Overall the topology of the represented Croton clades or sections (Fig. 5) was congruent with previous Croton phylogenetic analyses (Berry et al. 2005

Distribution, ecology, and phenology
Croton maranonensis sp.nov. is distributed in the Marañón Valley in northern Peru and in the Province of Loja, southern Ecuador.According to information from specimens labels, the species grows in seasonally dry tropical forest and dry shrublands, along semideciduous shrubs from different plant families, giant columnar cacti, ferns, and epiphytic bromeliads (Fig. 6).Examined specimens were in flower and fruit throughout the year.

Preliminary conservation status assessment
The extent of occurrence (EOO) and area of occupancy (AOO) resulted in 26,335.574km 2 for EOO and 72,000 km 2 for AOO.Following the IUCN criteria (IUCN 2012), Croton maranonensis sp.nov.cannot be classified as vulnerable (VU) using the B1 criterion due to its disjunct distribution with relatively isolated populations in southern Ecuador and a few records (Gonzáles 6491 and Marcelo-Peña 11142,

Discussion
New species discovery, elaboration of morphological description, and the final publication of a new taxon is often a long process which could extend for decades (Bebber et al. 2010;Riina et al. 2015;Goodwin et al. 2020).This is the case of Croton maranonensis sp.nov., whose first specimen was collected in 1877, about 145 years ago, by the famous french botanist and explorer Édouard François André (Smith 1965;Padilla 1984).We provide strong evidence from multiple sources that support the recognition of this species as an independent Croton lineage within C. section Julocroton.Both morphological (including anatomy) and molecular data provide strong support for establishing C. maranonensis  We observed slight morphological differences between the Peruvian and Ecuadorian populations of C. maranonensis sp.nov., such as the presence of sparse, dark ferrugineous, stellate trichomes (interspersed with withish trichomes) on the apical parts of branches and inflorescences and the more costate stems in the Ecuadorian specimens.However, scattered ferrugineous stellate trichomes were also observed in some specimens from Peru, especially on young leaves and young inflorescences.
Croton maranonensis sp.nov.might be the undetermined Croton species (as 'Croton sp.') reported by Marcelo-Peña et al. (2016) in their study of the Marañón flora.However, the authors did not include voucher information that could allow us to verify the species identity.Similarly, researchers conducting ecological studies in the southern Ecuadorian dry shrublands, where C. maranonensis sp.nov.could occur, were unable to identify the focal Croton taxa under study because of incomplete taxonomic knowledge of the species dominating those dry habitats and the possibility of hybridization involving populations of closely related species (Vélez-Mora et al. 2022).Additional systematics studies, including a phylogeographic approach, are needed to understand species boundaries among these abundant Croton shrubs from arid Andean ecosystems (e.g., Luján et al. 2015).
In our dissections of specimens of C. flavispicatus and C. maranonensis sp.nov.flowers, we found that style branches are very fragile and they are often broken in herbarium specimens.This may lead to confusion when this character is used to separate these species.Morphological differences between C. triqueter, C. hondensis, and C. flavispicatus are very subtle, which is also reflected in the results of the phylogenetic analysis, where specimens from the three species are embedded in a polytomy.A specimen of C. conspurcatus is also part of this polytomy but is well resolved in a clade with one of the accessions of C. triqueter (accession 4).These preliminary phylogenetic results indicate that further phylogenetic studies using a wider taxon sampling of section Julocroton and related clades will be needed to better establish species limits within this understudied section of Neotropical Croton.
The presence of the acropetiolar/basilaminar clusters of colleters (Figs 1B, 3I) is a novel finding for the genus Croton as a whole.So far, this feature is only known for C. maranonensis sp.nov., but it is possible that it could be present in other Julocroton species.Interestingly, these raceme of petiolar colleters are situated in the usual position of nectary glands in other Croton sections.However, it is unlikely that these colleters have the same function (ant attraction) of extrafloral nectaries in Croton (Aguirre et al. 2013;Leal et al. 2015) since colleters are only active in their early stage of leaf development (Vitarelli et al. 2015;Meira et al. 2020).
Similar to other Croton species that occur in dry environments (Vitarelli et al. 2015(Vitarelli et al. , 2021;;Sodré et al. 2019), C. maranonensis sp.nov.bears sclereids associated with epidermal trichomes in leaves.Therefore, our results reinforce previous findings about the functional role of these structures as capable of atmospheric water absorption, which has been suggested as a survival strategy for Croton species that live in environments with strong soil water restrictions (Vitarelli et al. 2016(Vitarelli et al. , 2021)).In fact, C. maranonensis sp.nov.occurs in extreme dry habitats, subject to a severe dry season (8-9 months) with an average annual rainfall of ca 550 mm in the Marañón Valley (Marcelo-Peña et al. 2016) and 317 mm in the southern Ecuadorian inter-Andean valleys (Vélez-Mora et al. 2022).
Regarding the suggested conservation status for the new species, it will be important to explore possible under-collected areas within the two gaps, in Peru and Ecuador, shown in the distribution map (Fig. 6), since there are areas of dry forest in those areas (DRYFLOR 2016;Quintana et al. 2017) where C. maranonensis sp.nov.populations could be present.

Fig. 1 .
Fig. 1.Croton maranonensis sp.nov. A. Image of the holotype: MA[874921].B. Clusters of no longer active colleters at the base of the lamina.C. Detail of the stipules.D. Leaf apex bearing a colleter (no longer active) at the tip.E. Seeds in different views.F. Inflorescences showing mainly the staminate portion (distal) with flowers in bud.G. Individual staminate flower.H. Detail of the calyx showing the three conspicuous and dissected lobes and the two highly reduced ones.I. Detail of the fruit showing the bifid styles.A from R. Riina & J. Campos 1469 (MA); B, E from van der Werff 14623 (MICH); C from Rojas 0863 (WIS); D, F. from van der Werff 16399 (WIS); G from Campos 2139 (MICH, MO); H-I from Cumbicus & Camacho 1924.Scale bars: B, D = 500 µm; C, F = 2 mm; E, G-I = 1 mm.

Fig. 2 .
Fig. 2. Croton maranonensis sp.nov. A. Small individual growing in dry forest understory.B. Fertile branch.C. Inflorescence showing both pistillate and staminate flowers; note the conspicuous colleter at the apex of the leaf on the right.D. Detail of inflorescence with two staminate flowers in anthesis.E. A view of the open dry vegetation associated with the seasonally dry tropical forest in the Marañón Valley where the new species occur.A-B from R. Riina 1478; C from Cumbicus 1924; D from Gonzáles 6491.Photographs by R. Riina (A-B), N. Cumbicus (C), and P. Gonzáles (D-E).
; Van Ee et al. 2011).Croton sect.Julocroton, C. sect.Lasiogyne (Klotzsch) Baill.and C. sect.Heptallon (Raf.)Müll.Arg.were recovered as highly supported monophyletic groups (Posterior Probability, PP = 1).The three specimens of C. maranonensis sp.nov.were clustered together in a highly supported clade (PP = 1) within the C. sect.Julocroton clade.The new species was recovered sister to a clade including C. triqueter, C. flavispicatus, C. hondensis, C. conspurcatus Schltdl.and C. fuscecens Spreng., albeit with relatively low support (PP = 0.79).The three accessions of C. hondensis and the two specimens of C. fuscescens were recovered as monophyletic groups, while the four representatives of C. triqueter emerged as unresolved within the largest clade including C. flavispicatus, C. hondensis, C. triqueter, and C. conspurcatus.The clade including C. argenteus was recovered sister to the rest of the C. sect.Julocroton.

Fig. 5 .
Fig. 5. Bayesian phylogram based on ITS sequences of the selected Croton species including C. maranonensis sp.nov.and representatives of sections Julocroton, Lasiogyne, Heptallon, and other sections of Croton.Numbers at nodes indicate Bayesian posterior probability values (PP).Numbers distinguishing accessions of the same species are also indicated in Appendix 2. The country of origin of the sample is indicated only for the samples of the new species..

Fig. 6 .
Fig. 6.Map showing the location of the known herbarium collections of Croton maranonensis sp.nov.(solid circles).Main cities in the area are indicated by white triangles.